Modified forms of pseudomonas exotoxin a

ABSTRACT

Pseudomonas  exotoxin A or “PE” is a 66 kD, highly potent, cytotoxic protein secreted by the bacterium  Pseudomonas aeruginosa . Various forms of PE have been coupled to other proteins, such as antibodies, to generate therapeutically useful cytotoxin conjugates that selectively target cells of a desired phenotype (such as tumor cells). In the present invention, peptides spanning the sequence of an approximately 38 kD form of  Pseudomonas  exotoxin A protein were analyzed for the presence of immunogenic CD4+ T cell epitopes. Six immunogenic T cell epitopes were identified. Residues were identified within each epitope for introduction of targeted amino acid substitutions to reduce or prevent immunogenic T-cell responses in PE molecules which may be administered to a heterologous host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. No.61/531,576, filed

Reference to Related Application

This application claims benefit of and priority based on U.S.Provisional Patent Application Ser. No. 61/531,576, filed Sep. 6, 2011,the contents of which are herein incorporated by reference in theirentirety. Sep. 6, 2011 which is herein incorporated by reference in itsentirety.

NAMES OF THE PARTIES IN A JOINT RESEARCH AGREEMENT

The claimed invention was made pursuant to a joint research agreement,as defined in 35 U.S.C. §103 (c)(3), that was in effect on or before thedate the claimed invention was made, and as a result of activitiesundertaken within the scope of the joint research agreement, by or onbehalf of the Intrexon Corp. (Foster City, Calif., U.S.A.) and AntitopeLtd. (Cambridge, UK).

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A Sequence Listing is submitted electronically via EFS-Web as an ASCIIformatted sequence listing in a file named “OT050-PCT_SEQLIST.txt”,created on Sep. 4, 2012, and having a file size of 295,678 bytes whichis filed concurrently with the present specification, claims, abstractand figures provided herewith. The sequence listing contained in thisASCII formatted document is part of the specification and is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Immune System and T Cell Epitopes

Immune responses to biological therapeutic agents are wide ranging, andcan be directed against agents that are both non-human and human inorigin. These responses include those that elicit a weak clinical effectand those that limit efficacy which can occasionally result in morbidityor even mortality in patients. In particular, serious complications canarise with the production of neutralizing antibodies, especially whenthey target recombinant self proteins and therefore have the potentialto cross react with the patient's own endogenous protein (Lim, 2005).Problems associated with immunogenicity to biologics (i.e., therapeuticmedical products; such as, antibodies and recombinantproteins/polypeptides) have been reduced largely due to advances inmolecular biology. There are, however, many recombinant proteinbiologics that are identical to endogenously expressed human sequencesthat still elicit potent neutralizing immune responses in patients(Hochuli, 1997; Schellekens et al, 1997; Namaka et al, 2006). Themechanism by which immunogenicity is triggered remains unclear althoughthe tolerance to self proteins may be broken by a number of factorslinked to both the product and the patient (reviewed in Chester et al,2006; Baker and Jones, 2007). For the product, these include dose,frequency of administration, route, immunomodulatory capacity of theprotein therapeutic, and the formulation (Jaber and Baker, 2007). Forthe patient, factors such as immune competence (i.e. whether the patientis receiving immunosuppressive treatment), patient's MHC haplotype andintrinsic tolerance to the protein therapeutic will influenceimmunogenicity. Regardless of how immunogenicity is triggered, one ofthe single most important factors in the development of an ensuingimmune response is the presence of epitopes that are able to effectivelystimulate a potent CD4+ T cell response (reviewed Baker and Jones,2007).

T cells or T lymphocytes are a subset of white blood cells known aslymphocytes. (The abbreviation “T” in T cell is for “thymus” since thisis the primary organ responsible for T cell maturation.) T cells play acentral role in cell-mediated immunity. They can be distinguished fromother types of lymphocytes (such as B cells and natural killer cells (NKcells)), by the presence of cell-surface proteins called T cellreceptors (TCRs). Different types of T cells have also been identified;these can be distinguished based on the differing functions they serve(e.g., CD4+ T cells (a.k.a., T_(H) or T helper cells), CD8+ cytotoxic Tcells (CTLs), memory T cells, regulatory T cells (T_(reg) cells),natural killer cells (NK cells), and gamma delta T cells (γδ T cells)).

T helper (T_(H)) cells are so named because they aid other white bloodcells in immunologic processes including, inter alia, assisting thematuration of B cells into plasma and B memory cells, and activation ofcytotoxic T cells and macrophages. T_(H) cells are also known as CD4+ Tcells because they express CD4 protein on the cell-surface. CD4+ T cellsare activated when peptide antigens are presented by MHC class IImolecules expressed on the surface of Antigen Presenting Cells (APCs).Once activated, CD4+ T cells divide rapidly and secrete chemokines thatfurther assist in activating or regulating immune responses.

T cell epitope analysis is becoming increasingly important particularlyin the pre-clinical analysis of biologics and may, in time, become arequirement for regulatory approval for clinical trials. To this end, apre-clinical ex vivo T cell assay (EPISCREEN™) has been used to providean effective technology for predicting T cell immunogenicity byidentifying linear T cell epitopes present in protein sequences.Synthetic overlapping peptides typically of about 15 amino acids inlength are tested against a cohort of community blood donors carefullyselected based on MHC class II haplotypes to provide a quantitativeanalysis of T cell epitopes present in protein sequences. Thistechnology has been used successfully to compare protein variants forthe potential to induce an immune response in vivo. By providing a highdegree of sensitivity along with high reproducibility, the EPISCREEN™assay allows an accurate pre-clinical assessment of the potential forimmunogenicity of biologics. See, Baker & Carr, “PreclinicalConsiderations in the Assessment of Immunogenicity for ProteinTherapeutics,” Current Drug Safety 5(4):1-6 (2010); Bryson et al.,“Prediction of Immunogenicity of Therapeutic Proteins: Validity ofComputational Tools,” Biodrugs 24(1)1-8 (2010); Holgate & Baker,“Circumventing Immunogenicity in the Development of TherapeuticAntibodies,” IDrugs 12(4):233-237 (2009); Perry et al., “New Approachesto Prediction of Immune Responses to Therapeutic Proteins duringPreclinical Development,” Drugs R D 9(6):385-396 (2008); and, Baker &Jones, “Identification and removal of immunogenicity in therapeuticproteins,” Current Opinion in Drug Discovery & Development 10(2):219-227(2007).

Pseudomonas Exotoxin A

Pseudomonas exotoxin A (PE-A) is a highly potent, 66 kD, cytotoxicprotein secreted by the bacterium Pseudomonas aeruginosa. PE-A causescell death by inhibiting protein synthesis in eukaryotic cells viainactivation of translation elongation factor 2 (EF-2), which ismediated by PE-A catalyzing ADP-ribosylation of EF-2 (i.e., transfer ofan ADP ribosyl moiety onto EF-2). PE-A typically produces death bycausing liver failure.

PE-A has at least three different structural domains responsible forvarious biological activities (FIG. 1). See e.g., Siegall et al.,Biochemistry, vol. 30, pp. 7154-7159 (1991); Theuer et al., Jour. Biol.Chem., vol. 267, no. 24, pp. 16872-16877 (1992); and, U.S. Pat. No.5,821,238. PE-A domain IA (amino acids 1-252 (see e.g., SEQ ID NO:133))is responsible for cell binding. Domain II (amino acids 253-364 (seee.g., SEQ ID NO: 133)) is responsible for translocation of PE-A into thecell cytosol. Domain III, the cytotoxic domain (amino acids 400-613 (seee.g., SEQ ID NO:133)), is responsible for ADP ribosylation of ElongationFactor 2 (EF2); which thereby inactivates EF2, subsequently causing celldeath. Additionally, a function for domain IB (amino acids 365-399 (SEQID NO:139)) has not been established. Indeed, it has been reported thatamino acids 365-380 (SEQ ID NO:138) within domain IB can be deletedwithout producing an identifiable a loss of function. See, Siegall etal., Biochemistry, vol. 30, pp. 7154-7159 (1991).

It has also been reported that PE-A may comprise any one of at leastthree different carboxy-terminal tails (FIG. 1); these appear to beessential for maintaining or recycling proteins into the endoplasmicreticulum. See, Theuer et al., J. Biol. Chem., vol. 267, no. 24, pp.16872-16877 (1992); Chaudhary et al., Proc. Natl. Acad. Sci. USA, vol.87, pp. 308-312 (1990); and, Seetharam et al., Jour. Biol. Chem., vol.266, 17376-17381 (1991). In particular, in correspondence with theexemplary sequence shown in FIG. 1 (SEQ ID NO: 133) these alternativecarboxy-terminal tails comprise amino acid sequences:

609 - REDLK - 613; (SEQ ID NO: 135) 609 - REDL - 612; (SEQ ID NO: 136)and 609 - KDEL - 612. (SEQ ID NO: 137)

Variants of PE-A, modified to lack the cell binding domain but coupledto heterologous cell-specific targeting molecules (e.g., antibodies),have been shown to have reduced levels of non-specific toxicity. Seee.g., U.S. Pat. No. 4,892,827.

Various forms of PE-A (e.g., truncated/deletion forms with molecularweights of ˜37 kD, 38 kD, 40 kD, et cetera) have been combined with anumber of growth factors, antibodies, and other proteins to generatecytotoxins which selectively target cells of a desired phenotype. See,for example:

-   Kreitman et al., “Recombinant immunotoxins and other therapies for    relapsed/refractory hairy cell leukemia,” Leuk. Lymphoma, Suppl.    2:82-86 (June-2011);-   Itoi et al., “Targeting of locus ceruleus noradrenergic neurons    expressing human interleukin-2 receptor α-subunit in transgenic mice    by a recombinant immunotoxin anti-Tac(Fv)-PE38,” J. Neurosci.,    31(16):6132-6139 (April-2011);-   Shapira et al., “An immunoconjugate of anti-CD24 and Pseudomonas    exotoxin selectively kills human colorectal tumors in mice,”    Gastroenterology, 140(3):935-946 (March-2011);-   Kuan et al., “Affinity-matured anti-glycoprotein NMB recombinant    immunotoxins targeting malignant gliomas and melanomas,” Int. J.    Cancer, 129(1):111-21 (July-2011);-   Hu et al., “Investigation of a plasmid containing a novel    immunotoxin VEGF165-PE38 gene for antiangiogenic therapy in a    malignant glioma model,” Int. J. Cancer, 127(9):2222-2229    (November-2010);-   Mareeva et al., “A novel composite immunotoxin that suppresses    rabies virus production by the infected cells,” J. Immunol. Methods,    353(1-2):78-86 (February-2010);-   Zielinski et al., “Affitoxin—a novel recombinant, HER2-specific,    anticancer agent for targeted therapy of HER2-positive tumors,” J.    Immunother. 32(8):817-825 (October-2009);-   Theuer et al., J. Biol. Chem., 267(24):16872-16877 (1992);-   Pastan et al., “Recombinant toxins for cancer treatment,” Science,    254:1173-1177 (1991);-   U.S. Pat. No. 5,821,238 (“Recombinant Pseudomonas Exotoxins with    Increased Activity”); and-   U.S. Pat. No. 4,892,827 (“Recombinant Pseudomonas Exotoxins:    Construction of an Active Immunotoxin with Low Side Effects”).

A significant disadvantage in using PE-A for treatment of disease,however, is that it is a foreign (non-self) protein being introducedinto a heterologous host (e.g., a human). Introduction of non-selfproteins into heterologous hosts commonly elicits host immune reactions,such as the generation of antibodies (“neutralizing antibodies”) orimmune cell reactions (e.g., cytotoxic T cell responses) which aredirected at eliminating the non-self protein (i.e., PE-A). Accordingly,it would be advantageous if elements of PE-A (PE-A epitopes) which arerecognized and targeted as “non-self” could be removed prior to use ofthis molecule as a therapeutic agent.

Deimmunization of PE

Some investigators have previously attempted to identify and removeimmunogenic determinants from PE-A (i.e., to “deimmunize” PE-A). See,for example:

-   Pastan et al., “Immunotoxins with decreased immunogenicity and    improved activity,” Leukemia and Lymphoma, 52(S2):87-90 (June-2011);-   Onda et al., “Recombinant immunotoxin against B-cell malignancies    with no immunogenicity in mice by removal of B-cell epitopes,” Proc.    Natl. Acad. Sci. USA, 108(14):5742-5747 (April-2011);-   Hansen et al., “A recombinant immunotoxin targeting CD22 with low    immunogenicity, low nonspecific toxicity, and high antitumor    activity in mice,” J. Immunother. 33(3):297-304 (April-2011);-   Stish et al., “Design and modification of EGF4KDEL 7Mut, a novel    bispecific ligand-directed toxin, with decreased immunogenicity and    potent anti-mesothelioma activity,” Br. J. Cancer, 101(7):1114-1123    (October-2009);-   Nagata et al., “Removal of B cell epitopes as a practical approach    for reducing the immunogenicity of foreign protein-based    therapeutics,” Adv. Drug Deliv. Rev., 61(11):977-985    (September-2009);-   Onda et al., “An immunotoxin with greatly reduced immunogenicity by    identification and removal of B cell epitopes,” Proc. Natl. Acad.    Sci. USA, 105(32):11311-11316 (August-2008); and-   Pastan et al, “Mutated Pseudomonas Exotoxins with Reduced    Antigenicity,” U.S. Patent Application No. 2009/0142341.

Despite progress in the area of deimmunization of PE-A, there remains aneed for the development of optimized, less immunogenic ornon-immunogenic, biologically active forms of this useful cytotoxin. Theinvention described herein addresses this need.

BRIEF SUMMARY OF THE INVENTION

Peptides spanning the sequence of an approximately 38 kD (predictedmolecular weight) form of Pseudomonas exotoxin A protein (SEQ ID NO:1)were analyzed for the presence of immunogenic CD4+ T cell epitopes. Atotal of 120 overlapping 15mer peptides spanning this sequence (SEQ IDNO: 1), but also including an amino terminal (Gly_(x5)-Ser)_(x2) linkersequence (SEQ ID NO:3) to produce a 359 amino acid Gly-Ser-PE38polypeptide sequence (SEQ ID NO:2), were tested against a cohort ofhealthy human donors. CD4+ T cell responses against individual peptideswere measured via proliferation assays. Assay data was used to compile aT cell epitope map of the PE38 sequence. Six immunogenic T cell epitopeswere identified. Residues were then identified within each of theseepitopes for use in targeted amino acid substitutions to reduce orprevent PE38-induced immunogenicity. Reduction or prevention of PEimmunogenicity should allow for multiple therapeutic administrations ofcytotoxic PE for use, for example, in the targeted destruction of cancercells in vivo (such as when administered as an immunoconjugate orcell-surface targeted fusion protein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Example of a Pseudomonas exotoxin A protein and domains whichmay be contained therein.

FIG. 2. Comparison of the frequency of donor allotypes expressed in theIEX01 study cohort (n=52) and the world population.

FIGS. 3A & 3B. CD4+ T cell epitope map of IEX01 PE38 sequence usingoverlapping 15mer peptides tested against 52 healthy donors. Thenon-adjusted (FIG. 3A) and adjusted (FIG. 3B) proliferation assay datafor the PE38 sequence is shown. Peptides inducing positive (SI≧2.00,p<0.05, including borderline responses) T cell proliferation responsesat a frequency above the background response rate (mean positive T cellresponses plus SD) contain T cell epitopes (dotted line indicates thebackground response threshold). KLH induced positive responses in(SI≧2.00, p<0.05) 75% of (non-adjusted) donors.

FIG. 4. Alignment of peptides 50, 52 and 53 showing the predicted HLA-DRcore 9mer binding register. Predicted core 9mer sequences are bracketedby p1 and p9 anchor residues. Peptides that stimulated positive T cellresponses in the adjusted data set are shown. Amino acid numbering(residues 153 and 161) correspond to SEQ ID NO:2 (PE38 of SEQ ID NO: 1plus amino-terminal linker GGGGGSGGGGGS (SEQ ID NO:3)).

FIG. 5. Alignment of peptides 65, 67 and 68 showing one predicted HLA-DRcore 9mer binding register. Predicted core 9mer sequences are bracketedby p1 and p9 anchor residues. Peptides that stimulated positive T cellresponses in the adjusted data set are shown. Amino acid numbering(residues 196 and 204) correspond to SEQ ID NO:2 (PE38 of SEQ ID NO:1plus amino-terminal linker GGGGGSGGGGGS (SEQ ID NO:3)).

FIG. 6. Alignment of peptides 81 and 82 showing the potential HLA-DRcore 9mer binding register. Predicted core 9mer sequences are bracketedby p1 and p9 anchor residues. Peptides that stimulated positive T cellresponses in the adjusted data set are shown. Amino acid numbering(residues 244 and 252) correspond to SEQ ID NO:2 (PE38 of SEQ ID NO:1plus amino-terminal linker GGGGGSGGGGGS (SEQ ID NO:3)).

FIG. 7. Alignment of peptides 110 and 111 showing a predicted HLA-DRcore 9mer binding register. Predicted core 9mer sequences are bracketedby p1 and p9 anchor residues. Peptides that stimulated positive T cellresponses in the adjusted data set are shown. Amino acid numbering(residues 333 and 341) correspond to SEQ ID NO:2 (PE38 of SEQ ID NO:1plus amino-terminal linker GGGGGSGGGGGS (SEQ ID NO:3)).

FIG. 8. Position of CD4+ T cell epitopes within the PE38 sequence. Tcell epitopes identified by EPISCREEN™ T cell epitope mapping are shownas shaded bars above the sequence. The frequency of donors responding(SI≧2.00, p<0.05) to each epitope are indicated by the shading of thebars; light grey <10%, mid grey 10-14%; dark grey >=14%. Numbersassigned to each individual donor (that responded to a correspondingepitope) are included within each shaded bar.

FIG. 9. In vivo Transcription/Translation (IVTT) shows that circularplasmid expression vector encoding PE38-IL2 fusion protein was slightlybetter at inhibiting Luciferase protein synthesis compared to linearizedplasmid encoding the same PE38-IL2 fusion protein.

FIG. 10. Luciferase activity measure in counts per second (CPS) in 111vitro Transcription/Translation (IVTT) assays of genes encoding eitherWild-Type (WT) PE or encoding amino acid substituted PE.

FIG. 11. Analysis of production of cytokines IL-2 and IL-6 stimulated inresponse to expression of genes encoding either Wild-Type (WT) PE orencoding amino acid substituted PE.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Descriptions

Unless specifically indicated otherwise, as used herein the term “PE” or“PE-A” is intended to indicate a polypeptide comprising a cytotoxicpolypeptide sequence derived from a wild-type or naturally occurringform of Pseudomonas aeruginosa exotoxin A protein. In addition tocytotoxic polypeptide sequences, PE polypeptides may comprise additionalnaturally occurring or heterologous polypeptide sequences. Additionalnaturally occurring polypeptide sequences may include sequences such asare found in full-length Pseudomonas exotoxin A protein, for example,amino acid sequences responsible for cytosolic translocation andcell-specific targeting (as discussed further herein)). Additionalheterologous polypeptide sequences may include sequences with which atleast a PE cytotoxic polypeptide is fused to impart additional functionsor properties. (For example, a PE cytotoxic polypeptide may be fused toantigen binding polypeptide sequences such as an scFv antibody.)Examples of sequences comprising a cytotoxic portion of PE can be foundin SEQ ID NO:1 and SEQ ID NO:4 spanning amino acid residues Phe-134 toLys-347. Examples of sequences comprising a cytotoxic portion of PE canalso be found in SEQ ID NO:133 and SEQ ID NO:134 spanning amino acidresidues Phe-400 to Lys-613.

As used herein in reference to PE, unless indicated otherwise, a“cytotoxic polypeptide” or “cytotoxic polypeptide sequence” is intendedto indicate a polypeptide (or portion thereof) which is capable ofinactivating translation elongation factor 2 (EF-2), mediatingADP-ribosylation of EF-2, inhibiting protein synthesis, or inducing celldeath. For example, it has been demonstrated that PE domain III,comprised of amino acid residues 400-613 of SEQ ID NO: 133, issufficient to mediate ADP-ribosylation of EF-2 and thereby cause celldeath. See, Theuer et al., J. Biol. Chem., vol. 267, no. 24, pp.16872-16877 (1992) and Hwang et al., Cell, vol. 48, pp. 129-136 (1987).

Cytotoxic polypeptide sequences in the present invention may alsocomprise alternative carboxy-terminal sequences. See, Theuer et al.,Chaudhary et al. and, Seetharam et al. In particular embodiments,examples of carboxy-terminal tails of PE38 in the present invention maycomprise sequences as shown in FIG. 1 (SEQ ID NO:133). Hence, exemplaryalternative carboxy-terminal tails may comprise amino acid sequences:

(SEQ ID NO: 135; numbers 609-613 correspond to SEQ ID NO: 133)609 - REDLK - 613 (SEQ ID NO: 136; numbers 609-612 correspondto SEQ ID NO: 133) 609 - REDL - 612; and(SEQ ID NO: 137; numbers 609-612 correspond to SEQ ID NO: 133)609 - KDEL - 612.

Unless specifically indicated otherwise, as used herein the term “PE38”is intended to indicate a Pseudomonas aeruginosa exotoxin A (PE (orPE-A)) molecule comprising an amino acid sequence as shown in SEQ IDNO: 1. The amino acid sequence used to generate peptide sequencesreferenced in the Examples is shown in SEQ ID NO:2. SEQ ID NO:2comprises an amino terminal GGGGGSGGGGGS linker sequence (SEQ ID NO:3)fused to the PE38 amino acid sequence of SEQ ID NO: 1. A variant form ofPE38 is shown in SEQ ID NO:4. SEQ ID NO:4 differs from SEQ ID NO:1 bycomprising a Ser-to-Asn change at position 114, a Ile-to-Val change atposition 141, and a Gly-to-Ser change at position 249.

As used herein, unless specifically stated otherwise, “biologicalactivity” in reference to Pseudomonas exotoxin A (PE-A), PE or PE38 isintended to indicate at least one of the biological activities exhibitedby naturally occurring forms of the Pseudomonas aeruginosa exotoxin Amolecule. These activities include, for example, cell killing or cellcytotoxic activity (a.k.a., cell cytotoxicity), inactivation oftranslation elongation factor EF-2, ADP-ribosylation of EF-2, andinhibition of protein synthesis. The biological activity of PE and PE38polypeptides (and modified forms thereof; e.g., PE and PE38 amino acidsubstituted variants and fusion proteins) can be measured using assaysand experiments which are well-known and routinely used by those skilledin the art. Examples of some of these assays and experiments are furtherdescribed and referenced herein, without limitation, in the Examplessections included herein.

As used herein, the term “having Pseudomonas exotoxin A (PE-A)biological activity” (or “PE biological activity”) is intended toindicate molecules exhibiting about 5% or more of at least onebiological activity compared to a corresponding wild-type, naturallyoccurring, or non-amino acid substituted form of PE or PE-A. In someembodiments, molecules “having Pseudomonas exotoxin A biologicalactivity” (or “PE biological activity”) exhibit 5% or more, about 10% ormore, 10% or more, about 15% or more, 15% or more, about 20% or more,20% or more, about 25% or more, 25% or more, about 30% or more, 30% ormore, about 35% or more, 35% or more, about 40% or more, 40% or more,about 45% or more, 45% or more, about 50% or more, 50% or more, about60% or more, 60% or more, about 70% or more, 70% or more, about 75% ormore, 75% or more, about 80% or more, 80% or more, about 85% or more,85% or more, about 90% or more, 90% or more, about 95% or more, 95% ormore, about 100%, or 100% of at least one biological activity comparedto a corresponding wild-type, naturally occurring, or non-amino acidsubstituted forms of PE or PE-A.

As used herein, the term “wild-type” Pseudomonas exotoxin A (PE-A) (or“wild-type” PE) biological activity is intended to indicate at least oneor more biological activities exhibited by naturally occurring forms ofthe Pseudomonas exotoxin A (PE-A) or PE polypeptides. These include, forexample, without limitation, activities such as cell killing or cellcytotoxic activity (a.k.a., cell cytotoxicity), inactivation oftranslation elongation factor EF-2, ADP-ribosylation of EF-2, andinhibition of protein synthesis. Two examples, without limitation, ofpolypeptide sequences representing “wild-type” or non-amino acidsubstituted forms of PE-A are shown in SEQ ID NO: 133 and SEQ ID NO:134. Two examples, without limitation, of polypeptide sequencesrepresenting “wild-type” or non-amino acid substituted forms of PE areshown in SEQ ID NO:1 (PE38) and SEQ ID NO:4 (variant of PE38).

As used or claimed herein the term “a” or “an” in reference to thesubsequent recited entity refers to one or more of that entity; forexample, “a PE38 antibody” or “a polynucleotide encoding PE38” isunderstood to indicate one or more PE38 antibody molecules and one ormore polynucleotides encoding PE38, not a single PE38 antibody moleculenor a single polynucleotide molecule encoding PE38, respectively. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

Likewise, as used herein, the term “polypeptide” is intended toencompass a singular “polypeptide” as well as plural “polypeptides,” andrefers to a molecule composed of monomers (amino acids) linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, such as, but withoutlimitation glycosylation, acetylation, phosphorylation, amidation, etcetera. A “polypeptide” unless specifically described otherwise herein,may be derived from a natural biological source or produced byrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

Polypeptides may have a defined three-dimensional structure, althoughthey do not necessarily have such structure. Polypeptides with a definedthree-dimensional structure may be referred to as “folded” or having a“tertiary” structure. Polypeptides not configured into athree-dimensional structure, are referred to as unfolded. As usedherein, the term glycoprotein refers to a protein coupled to at leastone carbohydrate moiety attached to the protein via a covalent bond.

The term “isolated” is intended to indicate a biological component nolonger in its naturally occurring milieu. For example, an “isolatedpolypeptide” or “isolated polynucleotide” is intended to indicate apolypeptide or polynucleotide, respectively, which has been removed fromits naturally occurring milieu and which may have been inserted within anon-naturally occurring milieu. By way of example, this would include,without limitation, a polynucleotide which has been removed from anaturally occurring location within a host genome, and subsequentlyinserted, for example, into an expression vector or inserted into a newhost genome location or into the genome of a heterologous host organism.The “isolation” of a polypeptide or polynucleotide, as used herein,requires no particular level of purification. For example, recombinantlyproduced polypeptides expressed in host cells are considered isolatedfor purposes of the invention, as are native or recombinant polypeptideswhich have been separated, fractionated, or partially or substantiallypurified by any suitable technique.

Polypeptide embodiments also include fragments, derivatives, analogs,variants and fusion proteins; preferably but not necessarily whereinsuch embodiments retain one or more biological activities associatedwith a corresponding full-length or naturally occurring polypeptide.Fragments include proteolytic fragments, deletion fragments, andfragments encoded by synthetically or recombinantly producedpolynucleotides. Variants may occur naturally or be non-naturallyoccurring. Non-naturally occurring variants may be produced usingart-known mutagenesis techniques. Variant polypeptides may compriseconservative or non-conservative amino acid substitutions, deletions, oradditions. Derivatives include, but are not limited to, polypeptideswhich contain one or more non-naturally occurring amino acids,non-standard amino acids, and amino acid analogs. Polypeptideembodiments may comprise amino acid sequences which are at least 60%identical, at least 70% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 97%identical, at least 98% identical, or at least 99% identical to SEQ IDNO: 1.

Unless specifically defined otherwise, the term “polynucleotide” isintended to indicate nucleic acid molecules or constructs as routinelyused and understood by those of skill in the art. For example, nucleicacids include, but are not limited to, molecules such as messenger RNA(mRNA), plasmid DNA (pDNA), complementary DNA (cDNA), and genomic DNA(gDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The terms “polynucleotide” and “nucleicacid” are intended to include embodiments wherein any one or moresequences of polynucleotide or nucleic acid segments are contained, orcomprised within, a larger polynucleotide or nucleic acid sequence. Forexample, but without limitation, and unless stated otherwise to thecontrary herein, reference to a nucleic acid such as “a polynucleotideencoding PE38” is intended to include nucleic acids comprising “apolynucleotide encoding PE38” wherein such polynucleotide may also bepart of a larger nucleic acid or polynucleotide, such as an expressionvector or a polynucleotide/nucleic acid encoding an PE fusion protein.

An “isolated” nucleic acid or polynucleotide is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, a recombinant polynucleotide encoding anantibody contained in a vector is considered isolated for the purposesof the present invention. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) polynucleotides insolution. Isolated RNA molecules include in vivo or in vitro synthesizedRNA molecules; including synthetically produced molecules.

As used herein, a “coding region” is a portion of nucleic acidcontaining codons which may be translated into amino acids, although“stop codons” (TAG, TGA, or TAA) are not translated into an amino acids,but may also be considered to be part of a coding region. Unless statedotherwise herein, promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not considered part of a codingregion. Two or more coding regions of the present invention can bepresent in a single polynucleotide construct, e.g., on a single vector,or in separate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid embodiments may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding adifferent heterologous polypeptide. Heterologous coding regions includewithout limitation specialized elements or motifs, such as a secretorysignal peptide or a heterologous functional domains.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Othertranscription control elements, besides a promoter, include for example,but without limitation, enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

The terms “antibody” and “immunoglobulin” may be used interchangeablyherein. An antibody or immunoglobulin comprises at least theantigen-binding elements (e.g., complementarity determining regions orCDRs) of the variable domain of a heavy chain and/or of the variabledomain of a light chain. Basic immunoglobulin structures in vertebratesystems are well understood by those of skill in the art. See, e.g.,Harlow & Lane, Using Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 1999 (ISBN 0879695447)); see also, Harlow &Lane, Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988). The term “immunoglobulin” or “antibody” comprisesvarious broad classes of antibody molecules, such as, but withoutlimitation, IgG, IgM, IgA IgG, and IgE classes of antibodies; as well asantibody subclasses (isotypes), such as, IgG1, IgG2, IgG3, IgG4, IgA1,et cetera.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aVL or VH domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies.

As used herein, an “epitope” or “antigenic determinant” is the part of apolypeptide, antigen, or molecule that is recognized by the immunesystem, specifically by antibodies, B cells, or T cells. Epitopes ofpolypeptide antigens may function as conformational epitopes or linearepitopes. A conformational epitope is comprised of non-linear sectionsof a target molecule (such as that formed via the tertiary structure ofa folded polypeptide). In contrast, amino acids that make up a linearepitope may be comprised of a continuous sequence of amino acids or maybe comprised only of particular amino acid residues critical toantibody/B cell/T cell binding.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” may be used herein to qualify the relativeaffinity by which a certain antibody binds to a certain epitope. Forexample, antibody “A” may be deemed to have a higher specificity for agiven epitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)at pages 27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

The term “cross-reactivity” refers to the ability of an antibody,specific for one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

As used herein, the terms “linked,” “fused” or “fusion” may be usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two ore more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

A “variant” of a polypeptide or protein refers to any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature or may be generated artificially (e.g., viasynthetic or genetic engineering). Variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. Variants may include, inter alia:(a) variants in which one or more amino acid residues are substitutedwith, for example, conservative amino acids, non-conservative aminoacids, or amino acid analogs (b) variants in which one or more aminoacids are added to the polypeptide or protein, (c) variants in which oneor more of the amino acids includes a substituent group, and (d)variants in which the polypeptide or protein is fused with anotherpolypeptide such as serum albumin. The techniques for obtaining thesevariants, including genetic (suppressions, deletions, mutations, etc.),chemical, and enzymatic techniques, are known to those of skill in theart.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. It includeswithout limitation transcription of the gene into RNA molecules such as,for example, messenger RNA (mRNA), transfer RNA (tRNA) or any other RNAproduct, and the translation of mRNA into polypeptide(s).

Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, et cetera.

As used herein, the term “gene” refers to a polynucleotide comprisingnucleotides that encode a functional molecule, including functionalmolecules produced by transcription only (e.g., a bioactive RNA species)or by transcription and translation (e.g. a polypeptide). The term“gene” encompasses cDNA and genomic DNA nucleic acids. “Gene” alsorefers to a nucleic acid fragment that expresses a specific RNA, proteinor polypeptide, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and/or coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A chimeric gene may comprise coding sequences derived fromdifferent sources and/or regulatory sequences derived from differentsources. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene or“heterologous” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” refers to any genetic element(e.g., plasmid, phage, cosmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. The term “vector” includes both viral andnonviral vehicles for introducing the nucleic acid into a cell in vitro,ex vivo or in vivo. A large number of vectors known in the art may beused to manipulate nucleic acids, incorporate coding sequences intogenes, et cetera. Possible vectors include, for example, plasmids ormodified viruses including, for example bacteriophages such as lambdaderivatives, or plasmids such as pBR322 or pUC plasmid derivatives, orthe Bluescript vector. Another example of vectors that are useful in thepresent invention is the Ultra Vector™ Production System (IntrexonCorp., Blacksburg, Va.) as described in WO 2007/038276, incorporated byreference herein. For example, the insertion of the DNA fragmentscorresponding to response elements and promoters into a suitable vectorfor in vitro and/or in vivo expression of modified forms of PE (andfragments thereof) as described herein (including fusion proteins,conjugates, and otherwise linked forms) can be accomplished by ligatingthe appropriate DNA fragments into a chosen vector that hascomplementary cohesive termini. Alternatively, the ends of the DNAmolecules may be enzymatically modified or any site may be produced byligating nucleotide sequences (linkers) into the DNA termini. Suchvectors may be engineered to contain selectable marker genes thatprovide for the selection of cells that have incorporated the markerinto the cellular genome. Such markers allow identification and/orselection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used to express embodimentsof the invention described herein include, but are not limited to,retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpessimplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirusvectors. Non-viral vectors include plasmids, liposomes, electricallycharged lipids (cytofectins), DNA-protein complexes, and biopolymers. Inaddition to a nucleic acid, a vector may also comprise one or moreregulatory regions, and/or selectable markers useful in selecting,measuring, and monitoring nucleic acid transfer results (e.g.,monitoring transfer to target or non-target tissues, duration ofexpression, et cetera).

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid, preferably DNA, that replicates sequentially and whichcomprises an origin of replication, such as a plasmid, phage or cosmid,to which another nucleic acid segment may be attached so as to bringabout the replication of the attached segment. Cloning vectors may becapable of replication in one cell type and expression in another(“shuttle vector”). Cloning vectors may comprise one or more sequencesthat can be used for selection of cells comprising the vector and/or oneor more multiple cloning sites for insertion of sequences of interest.The term “expression vector” refers to a vector, plasmid or vehicledesigned to enable the expression of an inserted nucleic acid sequencefollowing transformation into the host. The cloned gene, i.e., theinserted nucleic acid sequence, is usually placed under the control ofcontrol elements such as a promoter, a minimal promoter, an enhancer, orthe like. Initiation control regions or promoters, which are useful todrive expression of a nucleic acid in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving expression of these genes can be used in an expressionvector, including but not limited to, viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoters, pathogenesis ordisease related promoters, developmental specific promoters, induciblepromoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL1O,ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TP1, alkalinephosphatase promoters (useful for expression in Saccharomyces); AOX1promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet,trp, IP_(L), IP_(R), T7, tac, and trc promoters (useful for expressionin Escherichia coli); light regulated-, seed specific-, pollenspecific-, ovary specific-, cauliflower mosaic virus 35S, CMV 35Sminimal, cassava vein mosaic virus (CsVMV), chlorophyll a/b bindingprotein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, rootspecific, chitinase, stress inducible, rice tungro bacilliform virus,plant super-promoter, potato leucine aminopeptidase, nitrate reductase,mannopine synthase, nopaline synthase, ubiquitin, zein protein, andanthocyanin promoters (useful for expression in plant cells); animal andmammalian promoters known in the art including, but are not limited to,the SV40 early (SV40e) promoter region, the promoter contained in the 3′long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters ofthe E1A or major late promoter (MLP) genes of adenoviruses (Ad), thecytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV)thymidine kinase (TK) promoter, a baculovirus IE1 promoter, anelongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase(PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, theregulatory sequences of the mouse metallothionein-L promoter andtranscriptional control regions, the ubiquitous promoters (HPRT,vimentin, α-actin, tubulin and the like), the promoters of theintermediate filaments (desmin, neurofilaments, keratin, GFAP, and thelike), the promoters of therapeutic genes (of the MDR, CFTR or factorVIII type, and the like), pathogenesis or disease related-promoters, andpromoters that exhibit tissue specificity and have been utilized intransgenic animals, such as the elastase I gene control region which isactive in pancreatic acinar cells; insulin gene control region active inpancreatic beta cells, immunoglobulin gene control region active inlymphoid cells, mouse mammary tumor virus control region active intesticular, breast, lymphoid and mast cells; albumin gene, Apo AI andApo A11 control regions active in liver, alpha-fetoprotein gene controlregion active in liver, alpha 1-antitrypsin gene control region activein the liver, beta-globin gene control region active in myeloid cells,myelin basic protein gene control region active in oligodendrocyte cellsin the brain, myosin light chain-2 gene control region active inskeletal muscle, and gonadotropic releasing hormone gene control regionactive in the hypothalamus, pyruvate kinase promoter, villin promoter,promoter of the fatty acid binding intestinal protein, promoter of thesmooth muscle cell α-actin, and the like. In addition, these expressionsequences may be modified by addition of enhancer or regulatorysequences and the like.

Vectors comprising polynucleotides of the invention may be introducedinto the desired host cells by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al, J. Biol. Chem. 267:963 (1992); Wu et al, J. Biol. Chem.263:14621 (1988); and Hartmut et al, Canadian Patent No. 2,012,311).

Vectors and polynucleotides of the invention may be introduced in vivoby lipofection. For example, via use of liposomes for encapsulation andtransfection of nucleic acids in vitro. Synthetic cationic lipidsdesigned to limit the difficulties encountered with liposome-mediatedtransfection can be used to prepare liposomes for in vivo transfectionof a gene encoding a marker (Feigner et al, Proc. Natl. Acad. Sci USA.84:7413 (1987); Mackey et al, Proc. Natl. Acad. Sci USA 85:8027 (1988);and Ulmer et al, Science 259:1745 (1993)). Use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Feigner et al,Science 337:387 (1989)). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described in WO95/18863,WO96/17823 and U.S. Pat. No. 5,459,127.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther. 3:147 (1992); and Wu et al., J. Biol. Chem.262:4429 (1987)).

The term “transfection” refers to the uptake of exogenous orheterologous RNA or DNA by a cell. A cell has been “transfected” byexogenous or heterologous RNA or DNA when such RNA or DNA has beenintroduced inside the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

In addition, recombinant vector comprising polynucleotides of theinvention may include one or more origins for replication in thecellular hosts in which their amplification or their expression issought, markers or selectable markers.

The term “selectable marker” refers to an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” refers to a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters may be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters.” Promoters that cause agene to be expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The promoter sequence is typically bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains nitrons) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” refer to DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

The term “response element” (“RE”) refers to one or more cis-acting DNAelements which confer responsiveness on a promoter mediated throughinteraction with the DNA-binding domains of a transcription factor. ThisDNA element may be either palindromic (perfect or imperfect) in itssequence or composed of sequence motifs or half sites separated by avariable number of nucleotides. The half sites can be similar oridentical and arranged as either direct or inverted repeats or as asingle half site or multimers of adjacent half sites in tandem. Theresponse element may comprise a minimal promoter isolated from differentorganisms depending upon the nature of the cell or organism into whichthe response element will be incorporated. The DNA binding domain of thetranscription factor binds, in the presence or absence of a ligand, tothe DNA sequence of a response element to initiate or suppresstranscription of downstream gene(s) under the regulation of thisresponse element. Examples of DNA sequences for response elements of thenatural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 140)(see Cherbas et. al., Genes Dev. 5:120-131 (1991)); AGGTCAN(n)AGGTCA(SEQ ID NO: 141), where N(n) can be one or more spacer nucleotides (seeD'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); andGGGTTGAATGAATTT (SEQ ID NO: 142) (see Antoniewski et al., Mol. CellBiol. 14:4465 (1994)).

The terms “operably linked,” “operably associated,” “through operableassociation,” and the like refer to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The terms “cassette,” “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of expressing polynucleotides and polypeptides undercontrol of a gene switch mechanism, the term “gene switch” refers to thecombination of a response element associated with a promoter, and aligand-dependent transcription factor-based system which, in thepresence of one or more ligands, modulates the expression of a gene intowhich the response element and promoter are incorporated. Statedotherwise, a “gene switch” refers to a peptide, protein or polypeptidecomplex that functions to (a) bind an activating ligand, and (b)regulate the transcription of a gene of interest in a ligand-dependentfashion.

As used herein with respect to gene switch regulation systems, the term“dimerizes with the ligand binding domain that binds an activatingligand” refers to a selective protein-protein interaction that isinduced by the presence of activating ligand.

As used herein, the term “ligand binding domain that binds an activatingligand” refers to an amino acid sequence that selectively binds anactivating ligand. In the methods disclosed herein, an activating ligandbinds to a ligand binding domain, e.g., an ecdysone receptor ligandbinding domain, that is part of a ligand-dependent transcriptionalactivation complex that regulates the expression of a polynucleotidesequence that encodes a gene of interest. Hence, the expression of thegene of interest is regulated in a ligand-dependent fashion.

The term “ecdysone receptor-based,” with respect to a gene switch,refers to a gene switch comprising at least a functional part of anaturally occurring or synthetic ecdysone receptor ligand binding domainand which regulates gene expression in response to a ligand that bindsto the ecdysone receptor ligand binding domain.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

Polynucleotides or vectors comprising sequences encoding polypeptides ofthe present invention may further comprise at least one promotersuitable for driving expression of a gene in a modified cell.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, et cetera.

“Regulatory region” refers to a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

The term “exogenous gene” or “heterologous gene” means a gene foreign tothe subject or organism, that is, a gene which is introduced into thesubject through a transformation process, an unmutated version of anendogenous mutated gene or a mutated version of an endogenous unmutatedgene. The method of transformation is not critical to this invention andmay be any method suitable for the subject known to those in the art.Exogenous genes can be either natural or synthetic genes which areintroduced into the subject in the form of DNA or RNA which may functionthrough a DNA intermediate such as by reverse transcriptase. Such genescan be introduced into target cells, directly introduced into thesubject, or indirectly introduced by the transfer of transformed cellsinto the subject.

Polynucleotides and polypeptides of the invention may be expressed invivo under control of a “gene switch” control mechanism, such as thosedescribed in, for example, but not limited to:

-   WO 2009/045370 (PCT/US2008/011270);-   WO 2009/025866 (PCT/US2008/010040);-   WO 2008/073154 (PCT/US2007/016747);-   WO 2005/108617 (PCT/US2005/015089);-   WO 2003/0/27289 (PCT/US2002/005026);-   WO 2002/066615 (PCT/US2002/005708);-   WO 2003/027266 (PCT/US/2002/05234);-   WO 2002/066612 (PCT/US2002/005090);-   WO 2002/066614 (PCT/US/2002/005706);-   WO 2002/066613 (PCT/US2002/005090);-   WO 2002/029075 (PCT/US2001/030608); and-   WO 2001/070816 (PCT/US2001/090500),    each of which are incorporated by reference herein.

The term “ligand-dependent transcription factor complex” or “LDTFC”refers to a transcription factor comprising one or more proteinsubunits, which complex can regulate gene expression driven by a“factor-regulated promoter” as defined herein. A model LDTFC is an“ecdysone receptor complex” generally refers to a heterodimeric proteincomplex having at least two members of the nuclear receptor family,ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins (see Yao etal., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)). Afunctional LDTFC such as an EcR complex may also include additionalprotein(s) such as immunophilins. Additional members of the nuclearreceptor family of proteins, known as transcriptional factors (such asDHR38, betaFTZ-1 or other insect homologs), may also be ligand dependentor independent partners for EcR and/or USP. A LDTFC such as an EcRcomplex can also be a heterodimer of EcR protein and the vertebratehomolog of ultraspiracle protein, retinoic acid-X-receptor (“RXR”)protein or a chimera of USP and RXR. The terms “LDTFC” and “EcR complex”also encompass homodimer complexes of the EcR protein or USP, as well assingle polypeptides or trimers, tetramer, and other multimers servingthe same function.

A LDTFC such as an EcR complex can be activated by an active ecdysteroidor non-steroidal ligand bound to one of the proteins of the complex,inclusive of EcR, but not excluding other proteins of the complex. Asused herein, the term “ligand,” as applied to LDTFC-based gene switchese.g., EcD complex based gene switches, describes small and solublemolecules having the capability of activating a gene switch to stimulateexpression of a polypeptide encoded therein. Examples of ligandsinclude, without limitation, an ecdysteroid, such as ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, and the like,9-cis-retinoic acid, synthetic analogs of retinoic acid,N,N′-diarylhydrazines such as those disclosed in U.S. Pat. Nos.6,013,836; 5,117,057; 5,530,028; 5,378,726; and 7,304,161 and U.S. Pat.No. 7,456,315; oxadiazolines as described in U.S. Pat. No. 7,304,162;dibenzoylalkyl cyanohydrazines such as those disclosed in EuropeanPatent No. 461,809B1; N-alkyl-N,N′-diarylhydrazines such as thosedisclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazinessuch as those disclosed in European Patent No. 234,994B1;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; amidoketones such as those described in U.S. Pat. No.7,375,093; each of which is incorporated herein by reference and othersimilar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonateesters, juvenile hormone III, and the like. Examples of diacylhydrazineligands useful in the present invention include RG-115819(3,5-Dimethyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide),RG-115932 ((R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), andRG-115830 (3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See,e.g., U.S. Pat. No. 8,076,517 (Publication No. 2009/0163592), and PCTAppl. No. PCT/US2008/006757 (WO 2008/153801), both of which areincorporated herein by reference in their entireties.

A LDTFC such as an EcR complex includes proteins which are members ofthe nuclear receptor superfamily wherein all members are characterizedby the presence of one or more polypeptide subunits comprising anamino-terminal transactivation domain (“AD,” “TD,” or “TA,” usedinterchangeably herein), a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”). The AD may be present as a fusion with a“heterodimerization partner” or “HP.” A fusion protein comprising an ADand HP of the invention is referred to herein as a “coactivationprotein” or “CAP.” The DBD and LBD may be expressed as a fusion protein,referred to herein as a “ligand-inducible transcription factor (“LTF”).The fusion partners may be separated by a linker, e.g., a hinge region.Some members of the LTF family may also have another transactivationdomain on the carboxy-terminal side of the LBD. The DBD is characterizedby the presence of two cysteine zinc fingers between which are two aminoacid motifs, the P-box and the D-box, which confer specificity forecdysone response elements. These domains may be either native,modified, or chimeras of different domains of heterologous receptorproteins.

EcR ligands, when used with a LDTFC, e.g., an EcR complex, which in turnis bound to the response element linked to an exogenous gene (e.g., areporter gene), provide the means for external temporal regulation ofexpression of the exogenous gene. The order in which the variouscomponents bind to each other, that is, ligand to receptor complex andreceptor complex to response element, is not critical. Typically,modulation of expression of the exogenous gene is in response to thebinding of a LDTFC, e.g., an EcR complex, to a specific control, orregulatory, DNA element. The EcR protein, like other members of thenuclear receptor family, possesses at least three domains, an AD, a DBD,and a LBD. This receptor, like a subset of the nuclear receptor family,also possesses less well-defined regions responsible forheterodimerization properties (referred to herein as a“heterodimerization partner” or “HP”). Binding of the ligand to theligand binding domain of a LTF, e.g., an EcR protein, afterheterodimerization with a CAP including, e.g., an AD and/or an HP, e.g.,a USP or RXR protein, enables the DNA binding domains of theheterodimeric proteins to bind to the response element in an activatedform, thus resulting in expression or suppression of the exogenous gene.This mechanism does not exclude the potential for ligand binding toindividual subunits, e.g., LTF or CAP, e.g., an EcR or USP, and theresulting formation of active homodimer complexes (e.g. EcR+EcR orUSP+USP). In one embodiment, one or more of the receptor domains can bevaried producing a chimeric gene switch. Typically, one or more of thethree domains may be chosen from a source different than the source ofthe other domains so that the chimeric receptor is optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) orLexA protein from E. coli (see Brent et al., Cell 43:729-736 (1985)) toaccommodate chimeric LDTFCs, e.g., EcR complexes. Another advantage ofchimeric systems is that they allow choice of a promoter used to drivethe exogenous gene according to a desired end result. Such doublecontrol can be particularly important in areas of gene therapy,especially when cytotoxic proteins are produced, because both the timingof expression as well as the cells wherein expression occurs can becontrolled. When exogenous genes, operatively linked to a suitablepromoter, are introduced into the cells of the subject, expression ofthe exogenous genes is controlled by the presence of the ligand of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcell) or specific to certain developmental stages of the organism.

For in vivo use, the ligands described herein may be taken up inpharmaceutically acceptable carriers, such as, for example, solutions,suspensions, tablets, capsules, ointments, elixirs, and injectablecompositions. Pharmaceutical compositions may contain from 0.01% to 99%by weight of the ligand. Compositions may be either in single ormultiple dose forms. The amount of ligand in any particularpharmaceutical composition will depend upon the effective dose, that is,the dose required to elicit the desired gene expression or suppression.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) and by naso-gastric tube. It willbe understood by those skilled in the art that the preferred route ofadministration will depend upon the condition being treated and may varywith factors such as the condition of the recipient.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development, progressionor spread (i.e., metastasis) of cancer. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal). “Treatment” can also mean prolonging survival as compared toexpected survival if not receiving treatment. Those in need of treatmentinclude those already with the condition or disorder as well as thoseprone to have the condition or disorder or those in which the conditionor disorder is to be prevented.

The terms “subject,” “individual,” “animal,” “patient,” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include, withoutlimitation, humans, domestic animals, farm animals, and zoo, sports, orpet animals such as dogs, cats, guinea pigs, rabbits, rats, mice,horses, cattle, cows, et cetera.

The terms “hyperproliferative disease or disorder” is intended toencompass all neoplastic cell growth and proliferation, whethermalignant or benign, including all transformed cells and tissues and allcancerous cells and tissues. Hyperproliferative diseases or disordersinclude, but are not limited to, precancerous lesions, abnormal cellgrowths, tumors (whether benign or malignant), “cancer” and otherhyperplasias.

The term “cancer” includes, but is not limited to, primary malignantcells or tumors (e.g., those whose cells have not migrated to sites inthe subject's body other than the site of the original malignancy ortumor) and secondary malignant cells or tumors (e.g., those arising frommetastasis, the migration of malignant cells or tumor cells to secondarysites that are different from the site of the original tumor).

A tumor or tumor tissue may also comprise “tumor-associated non-tumorcells”, e.g., vascular cells which form blood vessels to supply thetumor or tumor tissue. Non-tumor cells may be induced to replicate anddevelop by tumor cells, for example, the induction of angiogenesis in atumor or tumor tissue.

Some examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers are noted below and include:squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial cancer or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, as well as head (e.g., brain) and neck cancer.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma,Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult PrimaryLiver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma,AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer,Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, BreastCancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System(Primary) Lymphoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, ChildhoodBrain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood CerebralAstrocytoma, Childhood Extracranial Germ Cell Tumors, ChildhoodHodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamicand Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, ChildhoodMedulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal andSupratentorial Primitive Neuroectodermal Tumors, Childhood Primary LiverCancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,Childhood Visual Pathway and Hypothalamic Glioma, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T cellLymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma andRelated Tumors, Exocrine Pancreatic Cancer, Extracranial Germ CellTumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, EyeCancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer,Gastric Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalTumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin'sDisease, Hodgkin's Lymphoma, Hypergammaglobulinemia, HypopharyngealCancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, LaryngealCancer, Lip and Oral Cavity Cancer. Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer.Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

Naturally Occurring Amino Acid Substitutions

List of naturally occurring amino acids and some of their biochemicalproperties.

3- 1- Side-chain Letter Letter Side-chain charge Hydropathy Amino AcidCode Code polarity* (pH 7.4)* index** Alanine Ala A nonpolar neutral 1.8Arginine Arg R polar positive −4.5 Asparagine Asn N polar neutral −3.5Aspartic acid Asp D polar negative −3.5 Cysteine Cys C polar neutral 2.5Glutamic acid Glu E polar negative −3.5 Glutamine Gln Q polar neutral−3.5 Glycine Gly G nonpolar neutral −0.4 Histidine His H polarpositive(10%) −3.2 neutral(90%) Isoleucine Ile I nonpolar neutral 4.5Leucine Leu L nonpolar neutral 3.8 Lysine Lys K polar positive −3.9Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolarneutral 2.8 Proline Pro P nonpolar neutral −1.6 Serine Ser S polarneutral −0.8 Threonine Thr T polar neutral −0.7 Tryptophan Trp Wnonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine Val Vnonpolar neutral 4.2 *Hausman & Cooper, (2004), The Cell: A MolecularApproach, Washington, D.C: ASM Press. p. 51 (2004)( ISBN 0-87893-214-3).**Kyte & Doolittle, “A simple method for displaying the hydropathiccharacter of a protein,” Journal of Molecular Biology, 157(1): 105-132(May 1982).

Conservative Amino Acid Substitutions

Polypeptides may be made to differ by introduction of conservative ornon-conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similaramino acid side chains. “Conservative amino acid substitutions” refer tosubstitutions of one or more amino acids in a native amino acid sequence(e.g., wild-type or naturally occurring form of PE) with other aminoacid(s) having similar side chains (e.g., side chains similar in termsof size, charge, element composition, and/orhydrophobicity/hydrophilicity).

Conserved substitutes for an amino acid within a native amino acidsequence can be selected from other members of the group to which thenaturally occurring amino acid belongs. For example, conservative aminoacid residue substitution groups include:

(1) Alanine (A)-Glycine (G)-Serine (S)-Threonine;

(2) Aspartic acid (D)-Glutamic acid (E);

(3) Asparagine (N)-Glutamine (Q);

(4) Arginine (R)-Lysine (K)-Histidine (H);

(5) Isoleucine (I)-Leucine (L)-Methionine (M)-Valine (V); and

(6) Phenylalanine (F)-Tyrosine (Y)-Tryptophan (W).

Other substitution groups of amino acids can be envisioned. For example,amino acids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an Aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include:

-   -   Aromatic: Phenylalanine (F)-Tyrosine (Y)-Tryptophan (W);    -   Sulfur-containing: Methionine (M)-Cysteine (C);    -   Basic: Arginine (R)-Lysine (K)-Histidine (H);    -   Acidic: Aspartic acid (D)-Glutamic acid (E);    -   Non-polar uncharged residues: Cysteine (C)-Methionine        (M)-Proline (P); and    -   Hydrophilic Uncharged Residues: Serine (S)-Threonine        (T)-Asparagine (N)-Glutamine (Q).

Exemplary embodiments of conservative amino acid substitutions includethe interchangeability of: valine-leucine, valine-isoleucine-leucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, asparticacid-glutamic acid, and asparagine-glutamine.

Examples of Amino Acid Analogs and Non-Standard Amino Acid Residues

Examples of a few of the many possible amino acid analogs routinelyknown to those of skill in the art include, for example, but withoutlimitation, analogs such as: 4-hydroxyproline which may be substitutedfor proline; 5-hydroxylysine which may be substituted for lysine;3-methylhistidine which may be substituted for histidine; homoserinewhich may be substituted for serine; and ornithine which may besubstituted for lysine.

Examples of a few of the many possible non-standard amino acidsroutinely known to those of skill in the art include, for example, butwithout limitation, molecules such as: ornithine, citrulline,lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyricacid, β-alanine (3-aminopropanoic acid), selenocysteine and pyrrolysine.

Substitution mutations may be made by any technique for mutagenesisknown in the art including, for example, but not limited to, in vitrosite-directed mutagenesis (Hutchinson et al, J. Biol. Chem. 255:6551(1978); Zoller et al. DNA 3:479 (1984); Oliphant et al, Gene 44:177(1986); Hutchinson et al, Proc. Natl. Acad. Sci. USA 83:710 (1986)), useof TAB® linkers (Pharmacia), restriction endonuclease digestion/fragmentdeletion and substitution, PCR-mediated/oligonucleotide-directedmutagenesis, et cetera. PCR-based techniques are preferred forsite-directed mutagenesis (see Higuchi, 1989, “Using PCR to EngineerDNA”, in PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

Embodiments of the Invention

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

ISFSTRGTQ; (epitope 1; SEQ ID NO: 5) GTQNWTVER;(epitope 2; SEQ ID NO: 6) IVFGGVRAR; (epitope 3; SEQ ID NO: 7)ARSQDLDAI; (epitope 4; SEQ ID NO: 8) LRVYVPRSS;(epitope 5; SEQ ID NO: 9) and IPDKEQAIS (epitope 6; SEQ ID NO: 10)

-   -   wherein one or more amino acid residues in any one or more of        these epitopes are substituted with a different amino acid        residue.

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

(peptide 50 (epitope 1); SEQ ID NO: 60) GDGGDISFSTRGTQN;(peptide 52 (epitope 2); SEQ ID NO: 62) SFSTRGTQNWTVERL;(peptide 53 (epitope 2); SEQ ID NO: 63) TRGTQNWTVERLLQA;(peptide 65 (epitope 3); SEQ ID NO: 75) AQSIVFGGVRARSQD;(peptide 67 (epitope 4); SEQ ID NO: 77) GGVRARSQDLDAIWR;(peptide 68 (epitope 4); SEQ ID NO: 78) RARSQDLDAIWRGFY;(peptide 81 (epitope 5); SEQ ID NO: 91) NGALLRVYVPRSSLP;(peptide 82 (epitope 5); SEQ ID NO: 92) LLRVYVPRSSLPGFY;(peptide 110 (epitope 6); SEQ ID NO: 120) LDPSSIPDKEQAISA;(peptide 111 (epitope 6); SEQ ID NO: 121) SSIPDKEQAISALPD;(overlapping epitopes 1 and 2; SEQ ID NO: 131) ISFSTRGTQNWTVER;(overlapping epitopes 3 and 4; SEQ ID NO: 132) IVFGGVRARSQDLDAI

-   -   wherein one or more amino acid residues in any one or more of        these epitopes are substituted with a different amino acid        residue.

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

-   -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid residues at one        or more of positions 1, 6 and 9 are substituted with a different        amino acid residue;    -   b) GTQNWTVER (SEQ ID NO:6), wherein amino acid residues at one        or more of positions 3, 4 and 6 are substituted with a different        amino acid residue;    -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid residues at one        or more of positions 1 and 6 are substituted with a different        amino acid residue;    -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at one        or more of positions 4 and 7 are substituted with a different        amino acid residue;    -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid residues at one        or more of positions 1, 2 and 9 are substituted with a different        amino acid residue;    -   f) IPDKEQAIS (SEQ ID NO: 10), wherein amino acid residues at one        or more of positions 1, 4, 6 and 7 are substituted with a        different amino acid residue;    -   g) ISFSTRGTQNWTVER (SEQ ID NO: 131), wherein amino acid residues        at one or more of positions 1, 6, 9, 10 and 12 are substituted        with a different amino acid residue; and    -   h) IVFGGVRARSQDLDAI (SEQ ID NO: 132), wherein amino acid        residues at one or more of positions 1, 6, 11, and 14 are        substituted with a different amino acid residue.

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

-   -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid residues at one        or more of positions 1, 6 and 9 are substituted with a        conservative amino acid substitution;    -   b) GTQNWTVER (SEQ ID NO:6), wherein amino acid residues at one        or more of positions 3, 4 and 6 are substituted with a        conservative amino acid substitution;    -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid residues at one        or more of positions 1 and 6 are substituted with a conservative        amino acid substitution;    -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at one        or more of positions 4 and 7 are substituted with a conservative        amino acid substitution;    -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid residues at one        or more of positions 1, 2 and 9 are substituted with a        conservative amino acid substitution;    -   f) IPDKEQAIS (SEQ ID NO: 10), wherein amino acid residues at one        or more of positions 1, 4, 6 and 7 are substituted with a        conservative amino acid substitution;    -   g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein amino acid residues        at one or more of positions 1, 6, 9, 10 and 12 are substituted        with a conservative amino acid substitution; and    -   h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein amino acid residues        at one or more of positions 1, 6, 11, and 14 are substituted        with a conservative amino acid substitution.

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

-   -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid residues at one        or more of positions 1, 6 and 9 are substituted with a        conservative amino acid substitution;    -   b) GTQNWTVER (SEQ ID NO:6), wherein amino acid residues at one        or more of positions 3, 4 and 6 are substituted with a        conservative amino acid substitution;    -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid residues at one        or more of positions 1 and 6 are substituted with a conservative        amino acid substitution;    -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at one        or more of positions 4 and 7 are substituted with a conservative        amino acid substitution;    -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid residues at one        or more of positions 1, 2 and 9 are substituted with a        conservative amino acid substitution;    -   f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid residues at one        or more of positions 1, 4, 6 and 7 are substituted with a        conservative amino acid substitution;    -   g) ISFSTRGTQNWTVER (SEQ ID NO: 131), wherein amino acid residues        at one or more of positions 1, 6, 9, 10 and 12 are substituted        with a conservative amino acid substitution; and    -   h) IVFGGVRARSQDLDAI (SEQ ID NO: 132), wherein amino acid        residues at one or more of positions 1, 6, 11, and 14 are        substituted with a conservative amino acid substitution,    -   wherein the conservative amino acid substitution at one or more        of said positions in a) through f) is selected from the group        consisting of:    -   1) A is substituted with any one of G, I, L, S, T or V;    -   2) D is substituted with E;    -   3) I is substituted with any one of L, M or V;    -   4) K is substituted with any one of H or R;    -   5) L is substituted with any one of A, G, I, M or V;    -   6) N is substituted with any one of S, T or Q;    -   7) Q is substituted with any one of S, T or N;    -   8) R is substituted with any one of K or H;    -   9) S is substituted with any one of A, G, N, T or Q;    -   10) T is substituted with any one of A, G, N, Q or S; and    -   11) V is substituted with any one of A, G, I, L or M.

Embodiments of the invention also comprise or consist of isolatedpolypeptides and peptides comprising or consisting of theabove-referenced amino acids sequences, except wherein one or more aminoacids have been substituted with conservative amino acids substitutions.Embodiments of the invention further comprise or consist of isolatedpolypeptides (proteins) and peptides comprising or consisting of theabove-referenced amino acids sequences, except wherein one or more aminoacids have been substituted with amino acids which are naturallyoccurring, non-naturally occurring, non-standard amino acids, or aminoacid analogs.

Embodiments of the invention include isolated polypeptides (proteins)comprising or consisting of a modified form of Pseudomonas exotoxin A,or a fragment thereof, wherein said modified form, or fragment thereof,comprises an epitope selected from the group consisting of:

-   -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino the acid residue at        position 1 (I) is substituted with A, N, T, Q or H, or wherein        the amino acid residue at position 6 (R) is substituted with Q,        or wherein the amino acid residue at position 9 (Q) is        substituted with N or T, or wherein the amino acid sequence        ISFSTRGTQ (SEQ ID NO:5) comprises two or more of said        substitutions in any combination;    -   b) GTQNWTVER (SEQ ID NO:6), wherein the amino acid residue at        position 3 (Q) is substituted with N or T, wherein amino the        acid residue at position 4 (N) is substituted with K or R, or        wherein the amino acid residue at position 6 (T) is substituted        with K or R, or wherein the amino acid sequence GTQNWTVER (SEQ        ID NO:6) comprises two or more of said substitutions in any        combination;    -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino the acid residue at        position 1 (1) is substituted with A or N, or wherein the amino        acid residue at position 6 (V) is substituted with D, M, or N,        or wherein the amino acid sequence IVFGGVRAR (SEQ ID NO:7)        comprises substitutions at both positions in any combination of        amino acid residues A or N at position 1 (I) and D, M, or N at        position 6 (V);    -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino the acid residue at        position 4 (Q) is substituted with K or R, or wherein the amino        acid residue at position 7 (D) is substituted with K or R, or        wherein the amino acid sequence ARSQDLDAI (SEQ ID NO:8)        comprises substitutions with K or R in any combination at both        positions 4 (Q) and 7 (D);    -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino the acid residue at        position 1 (L) is substituted with A, or wherein the amino acid        residue at position 2 (R) is substituted with D, S or A, or        wherein the amino acid residue at position 9 (S) is substituted        with D, E, N, K, P or T, or wherein the amino acid sequence        LRVYVPRSS (SEQ ID NO:9) comprises two or more of said        substitutions in any combination;    -   f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid residues at one        or more of positions 1, 4, 6 and 7 are substituted with a        different amino acid residue, wherein amino the acid residue at        position 1 (I) is substituted with A, N, T, Q or H, or wherein        the amino acid residue at position 4 (K) is substituted with T,        or wherein the amino acid residue at position 6 (Q) is        substituted with D, or wherein the amino acid residue at        position 7 (A) is substituted with D, or wherein the amino acid        sequence IPDKEQAIS (SEQ ID NO:10) comprises two or more of said        substitutions in any combination;    -   g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein amino acid residues        at one or more of positions 1, 6, 9, 10 and 12 are substituted        with a different amino acid residues wherein amino the acid        residue at position 1 (I) is substituted with A, N, T, Q or H,        or wherein the amino acid residue at position 6 (R) is        substituted with Q, or wherein the amino acid residue at        position 9 (Q) is substituted with N or T, or wherein amino the        acid residue at position 10 (N) is substituted with K or R, or        wherein the amino acid residue at position 12 (T) is substituted        with K or R, or wherein the amino acid sequence ISFSTRGTQNWTVER        (SEQ ID NO: 131) comprises two or more of said substitutions in        any combination; and    -   h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein amino the acid        residue at position 1 (I) is substituted with A or N, or wherein        the amino acid residue at position 6 (V) is substituted with D,        M, or N, wherein amino the acid residue at position 11 (Q) is        substituted with K or R, or wherein the amino acid residue at        position 14 (D) is substituted with K or R, or wherein the amino        acid sequence IVFGGVRARSQDLDAI (SEQ ID NO:132) comprises two or        more of said substitutions in any combination.

Embodiments of the invention include an isolated polypeptide comprisinga modified form of Pseudomonas exotoxin A, or a fragment thereof,wherein said modified form, or fragment thereof, comprises one or moreamino acid substitutions selected from the group consisting of:

a) I at position 141 changed to any amino acid residue; (epitope 1)

b) R at position 146 changed to any amino acid residue; (epitope 1)

c) Q at position 149 changed to any amino acid residue; (epitope 1)

d) N at position 150 changed to any amino acid residue; (epitope 2)

e) T at position 152 changed to any amino acid residue; (epitope 2)

f) I at position 184 changed to any amino acid residue; (epitope 3)

g) V at position 189 changed to any amino acid residue; (epitope 3)

h) Q at position 194 changed to any amino acid residue; (epitope 4)

i) D at position 197 changed to any amino acid residue; (epitope 4)

j) L at position 233 changed to any amino acid residue; (epitope 5)

k) R at position 234 changed to any amino acid residue; (epitope 5)

l) S at position 241 changed to any amino acid residue; (epitope 5)

m) I at position 321 changed to any amino acid residue; (epitope 6)

n) K at position 324 changed to any amino acid residue; (epitope 6)

o) Q at position 326 changed to any amino acid residue; (epitope 6)

p) A at position 327 changed to any amino acid residue; (epitope 6)

q) any combination of one or more of a) through ao),

wherein the amino acid numbering corresponds to SEQ ID NO: 1.

Embodiments of the invention include an isolated polypeptide comprisinga modified form of Pseudomonas exotoxin A, or a fragment thereof,wherein said modified form, or fragment thereof, comprises one or moreamino acid substitutions selected from the group consisting of:

a) I at position 141 changed to A; (epitope 1)

b) I at position 141 changed to N; (epitope 1)

c) I at position 141 changed to T; (epitope 1)

d) I at position 141 changed to Q; (epitope 1)

e) I at position 141 changed to H; (epitope 1)

f) R at position 146 changed to Q; (epitope 1)

g) Q at position 149 changed to N; (epitope 1)

h) Q at position 149 changed to T; (epitope 1)

i) N at position 150 changed to R; (epitope 2)

j) N at position 150 changed to K; (epitope 2)

k) T at position 152 changed to R; (epitope 2)

l) T at position 152 changed to K; (epitope 2)

m) I at position 184 changed to A; (epitope 3)

n) I at position 184 changed to N; (epitope 3)

o) V at position 189 changed to D; (epitope 3)

p) V at position 189 changed to M; (epitope 3)

q) V at position 189 changed to N; (epitope 3)

r) Q at position 194 changed to R; (epitope 4)

s) Q at position 194 changed to K; (epitope 4)

t) D at position 197 changed to R; (epitope 4)

u) D at position 197 changed to K; (epitope 4)

v) L at position 233 changed to A; (epitope 5)

w) R at position 234 changed to D; (epitope 5)

x) R at position 234 changed to S; (epitope 5)

y) R at position 234 changed to A; (epitope 5)

z) S at position 241 changed to D; (epitope 5)

ab) S at position 241 changed to E; (epitope 5)

ac) S at position 241 changed to N; (epitope 5)

ad) S at position 241 changed to K; (epitope 5)

ae) S at position 241 changed to P; (epitope 5)

af) S at position 241 changed to T; (epitope 5)

ag) I at position 321 changed to A; (epitope 6)

ah) I at position 321 changed to N; (epitope 6)

ai) I at position 321 changed to T; (epitope 6)

ak) I at position 321 changed to Q; (epitope 6)

al) I at position 321 changed to H; (epitope 6)

am) K at position 324 changed to T; (epitope 6)

an) Q at position 326 changed to D; (epitope 6)

ao) A at position 327 changed to D; (epitope 6)

ap) any combination of one or more of a) through ao),

wherein the amino acid numbering corresponds to SEQ ID NO: 1.

Embodiments of the invention comprise isolated polypeptides as describedabove, including polypeptides comprising amino acid substitutionsintroduced at each of amino acid positions 141, 146, 149, 150, 152, 184,189, 194, 197, 233, 234, 241, 321, 324, 326 and 327 (in comparison tothe amino acid sequence of SEQ ID NO: 1).

Embodiments of the invention include isolated polypeptides (proteins)and peptides comprising, or consisting of, the following amino acidsequences:

GGGGGSGGGGGSPEG; (peptide 1; SEQ ID NO: 11) GGSGGGGGSPEGGSL;(peptide 2; SEQ ID NO: 12) GGGGGSPEGGSLAAL; (peptide 3; SEQ ID NO: 13)GGSPEGGSLAALTAH; (peptide 4; SEQ ID NO: 14) PEGGSLAALTAHQAC;(peptide 5; SEQ ID NO: 15) GSLAALTAHQACHLP; (peptide 6; SEQ ID NO: 16)AALTAHQACHLPLET; (peptide 7; SEQ ID NO: 17) TAHQACHLPLETFTR;(peptide 8; SEQ ID NO: 18) QACHLPLETFTRHRQ; (peptide 9; SEQ ID NO: 19)HLPLETFTRHRQPRG; (peptide 10; SEQ ID NO: 20) LETFTRHRQPRGWEQ;(peptide 11; SEQ ID NO: 21) FTRHRQPRGWEQLEQ; (peptide 12; SEQ ID NO: 22)HRQPRGWEQLEQCGY; (peptide 13; SEQ ID NO: 23) PRGWEQLEQCGYPVQ;(peptide 14; SEQ ID NO: 24) WEQLEQCGYPVQRLV; (peptide 15; SEQ ID NO: 25)LEQCGYPVQRLVALY; (peptide 16; SEQ ID NO: 26) CGYPVQRLVALYLAA;(peptide 17; SEQ ID NO: 27) PVQRLVALYLAARLS; (peptide 18; SEQ ID NO: 28)RLVALYLAARLSWNQ; (peptide 19; SEQ ID NO: 29) ALYLAARLSWNQVDQ;(peptide 20; SEQ ID NO: 30) LAARLSWNQVDQVIR; (peptide 21; SEQ ID NO: 31)RLSWNQVDQVIRNAL; (peptide 22; SEQ ID NO: 32) WNQVDQVIRNALASP;(peptide 23; SEQ ID NO: 33) VDQVIRNALASPGSG; (peptide 24; SEQ ID NO: 34)VIRNALASPGSGGDL; (peptide 25; SEQ ID NO: 35) NALASPGSGGDLGEA;(peptide 26; SEQ ID NO: 36) ASPGSGGDLGEAIRE; (peptide 27; SEQ ID NO: 37)GSGGDLGESIREQPE; (peptide 28; SEQ ID NO: 38) GDLGEAIREQPEQAR;(peptide 29; SEQ ID NO: 39) GEAIREQPEQARLAL; (peptide 30; SEQ ID NO: 40)IREQPEQARLALTLA; (peptide 31; SEQ ID NO: 41) QPEQARLALTLAAAE;(peptide 32; SEQ ID NO: 42) QARLALTLAAAESER; (peptide 33; SEQ ID NO: 43)LALTLAAAESERFVR; (peptide 34; SEQ ID NO: 44) TLAAAESERFVRQGT;(peptide 35; SEQ ID NO: 45) AAESEREVRQGTGND; (peptide 36; SEQ ID NO: 46)SERFVRQGTGNDEAG; (peptide 37; SEQ ID NO: 47) FVRQGTGNDEAGAAS;(peptide 38; SEQ ID NO: 48) QGTGNDEAGAASGPA; (peptide 39; SEQ ID NO: 49)GNDEAGAASGPADSG; (peptide 40; SEQ ID NO: 50) EAGAASGPADSGDAL;(peptide 41; SEQ ID NO: 51) AASGPADSGDALLER; (peptide 42; SEQ ID NO: 52)GPADSGDALLERNYP; (peptide 43; SEQ ID NO: 53) DSGDALLERNYPTGA;(peptide 44; SEQ ID NO: 54) DALLERNYPTGAEFL; (peptide 45; SEQ ID NO: 55)LERNYPTGAEFLGDG; (peptide 46; SEQ ID NO: 56) NYPTGAEFLGDGGDI;(peptide 47; SEQ ID NO: 57) TGAEFLGDGGDISFS; (peptide 48; SEQ ID NO: 58)EFLGDGGDISFSTRG; (peptide 49; SEQ ID NO: 59) GDISFSTRGTQNWTV;(peptide 51; SEQ ID NO: 61) TQNWTVERLLQAHRQ; (peptide 54; SEQ ID NO: 64)WTVERLLQAHRQLEE; (peptide 55; SEQ ID NO: 65) ERLLQAHRQLEERGY;(peptide 56; SEQ ID NO: 66) LQAHRQLEERGYVFV; (peptide 57; SEQ ID NO: 67)HRQLEERGYVFVGYH; (peptide 58; SEQ ID NO: 68) LEERGYVFVGYHGTF;(peptide 59; SEQ ID NO: 69) RGYVFVGYHGTFLEA; (peptide 60; SEQ ID NO: 70)VFVGYHGTFLEAAQS; (peptide 61; SEQ ID NO: 71) GYHGTFLEAAQSIVF;(peptide 62; SEQ ID NO: 72) GTFLEAAQSIVFGGV; (peptide 63; SEQ ID NO: 73)LEAAQSIVFGGVRAR; (peptide 64; SEQ ID NO: 74) IVFGGVRARSQDLDA;(peptide 66; SEQ ID NO: 76) SQDLDAIWRGFYIAG; (peptide 69; SEQ ID NO: 79)LDAIWRGFYIAGDPA; (peptide 70; SEQ ID NO: 80) IWRGFYIAGDPALAY;(peptide 71; SEQ ID NO: 81) GFYIAGDPALAYGYA; (peptide 72; SEQ ID NO: 82)IAGDPALAYGYAQDQ; (peptide 73; SEQ ID NO: 83) DPALAYGYAQDQEPD;(peptide 74; SEQ ID NO: 84) LAYGYAQDQEPDARG; (peptide 75; SEQ ID NO: 85)GYAQDQEPDARGRIR; (peptide 76; SEQ ID NO: 86) QDQEPDARGRIRNGA;(peptide 77; SEQ ID NO: 87) EPDARGRIRNGALLR; (peptide 78; SEQ ID NO: 88)ARGRIRNGALLRVYV; (peptide 79; SEQ ID NO: 89) RIRNGALLIVYVPRS;(peptide 80; SEQ ID NO: 90) VYVPRSSLPGFYRTG; (peptide 83; SEQ ID NO: 93)PRSSLPGFYRTGLTL; (peptide 84; SEQ ID NO: 94) SLPGFYRTGLTLAAP;(peptide 85; SEQ ID NO: 95) GFYRTGLTLAAPEAA; (peptide 86; SEQ ID NO: 96)RTGLTLAAPEAAGEV; (peptide 87; SEQ ID NO: 97) LTLAAPEAAGEVERL;(peptide 88; SEQ ID NO: 98) AAPEAAGEVERLIGH; (peptide 89; SEQ ID NO: 99)EAAGEVERLIGHPLP; (peptide 90; SEQ ID NO: 100) GEVERLIGHPLPLRL;(peptide 91; SEQ ID NO: 101) ERLIGHPLPLRLDAI;(peptide 92; SEQ ID NO: 102) IGHPLPLRLDAITGP;(peptide 93; SEQ ID NO: 103) PLPLRLDAITGPEEE;(peptide 94; SEQ ID NO: 104) LRLDAITGPEEEGGR;(peptide 95; SEQ ID NO: 105) DAITGPEEEGGRLET;(peptide 96; SEQ ID NO: 106) TGPEEEGGRLETILG;(peptide 97; SEQ ID NO: 107) EEEGGRLETILGWPL;(peptide 98; SEQ ID NO: 108) GGRLETILGWPLAER;(peptide 99; SEQ ID NO: 109) LETILGWPLAERTVV;(peptide 100; SEQ ID NO: 110) ILGWPLAERTVVIPS;(peptide 101; SEQ ID NO: 111) WPLAERTVVIPSAIP;(peptide 102; SEQ ID NO: 112) AERTVVIPSAIPTDP;(peptide 103; SEQ ID NO: 113) TVVIPSAIPTDPRNV;(peptide 104; SEQ ID NO: 114) IPSAIPTDPRNVGGD;(peptide 105; SEQ ID NO: 115) AIPTDPRNVGGDLDP;(peptide 106; SEQ ID NO: 116) TDPRNVGGDLDPSSI;(peptide 107; SEQ ID NO: 117) RNVGGDLDPSSIPDK;(peptide 108; SEQ ID NO: 118) GGDLDPSSIPDKEQA;(peptide 109; SEQ ID NO: 119) PDKEQAISALPDYAS;(peptide 112; SEQ ID NO: 122) EQAISALPDYASQPG;(peptide 113; SEQ ID NO: 123) ISALPDYASQPGKPP;(peptide 114; SEQ ID NO: 124) LPDYASQPGKPPRED;(peptide 115; SEQ ID NO: 125) YASQPGKPPREDLK;(peptide 116; SEQ ID NO: 126) ITGPEEEGGRLDTIL;(peptide 117; SEQ ID NO: 127) PEEEGGRLDTILGWP;(peptide 118; SEQ ID NO: 128) EGGRLDTILGWPLAE;(peptide 119; SEQ ID NO: 129) and RLDTILGWPLAERTV.(peptide 120; SEQ ID NO: 130)

Embodiments of the invention also comprise or consist of isolatedpolypeptides (proteins) and peptides comprising or consisting of theabove-referenced amino acids sequences, except wherein one or more aminoacids have been substituted with conservative amino acids substitutions.Embodiments of the invention also comprise or consist of isolatedpolypeptides (proteins) and peptides comprising or consisting of theabove-referenced amino acids sequences, except wherein one or more aminoacids have been substituted with amino acids which are naturallyoccurring, non-naturally occurring, non-standard amino acids, or aminoacid analogs.

Embodiments of the invention include polypeptides comprising a PE-ADomain III (i.e., a cytotoxic domain; see e.g., FIG. 1). Examples ofsequences comprising a cytotoxic portion of PE can be found in SEQ IDNO:1 and SEQ ID NO:4 spanning amino acid residues Phe-134 to Lys-347.Examples of sequences comprising a cytotoxic portion of PE can also befound in SEQ ID NO:133 and SEQ ID NO:134 spanning amino acid residuesPhe-400 to Lys-613.

Embodiments of the invention include polypeptides comprising a PE-ADomain III (i.e., a cytotoxic domain) and one or more PE-A domainsselected from the group consisting of:

-   -   (a) Domain II (i.e., a cytosolic translocation domain; e.g.,        amino acids corresponding to Gly-3 to Ser-114 in SEQ ID NO:1 or        amino acids corresponding to Gly-3 to Asn-114 in SEQ ID NO:4,        see e.g., FIG. 1);    -   (b) Carboxy-terminal portion of Domain IB (e.g., amino acids        corresponding to Gly-115 to Glu-133 in SEQ ID NO:1 or SEQ ID        NO:4; and amino acids corresponding to Gly-381 to Glu-399 in SEQ        ID NO:133 or SEQ ID NO:134; see e.g., FIG. 1);    -   (c) Amino-terminal portion of Domain IB (e.g., amino acids        corresponding to Ala-365 to Ala-380 in SEQ ID NO:133 or SEQ ID        NO:134; see e.g., FIG. 1);    -   (d) Domain IB (i.e., amino acid sequences intervening between        Domains II and III; e.g., amino acids corresponding to Ala-365        to Glu-399 in SEQ ID NO:133 or SEQ ID NO:134; see e.g., FIG. 1);    -   (e) a carboxy-terminal tail selected from the group consisting        of:

(i) Arg-Glu-Asp-Leu-Lys; (SEQ ID NO: 135) (ii) Arg-Glu-Asp-Leu;(SEQ ID NO: 136) and (iii) Lys-Asp-Glu-Leu, (SEQ ID NO: 137)

-   -   wherein one or more of said domains has been modified with amino        acid substitutions, as described herein, to reduce or eliminate        immunogenicity.

Embodiments of the invention further comprise PE variants. For example,such variants include, without limitation, PE polypeptide examples asshown in SEQ ID Nos: 143 to 163 and SEQ ID No: 175.

Embodiments of the invention comprise any one or more of the PE-Adomains indicated in the preceding paragraphs, wherein said one or moredomains are chemically linked, covalently coupled, or fused (i.e., asin-frame fusion proteins) with a heterologous polypeptide (for example,such as ligand or antigen-binding polypeptide).

Embodiments of the invention include polypeptides comprising PE whereinone or more amino acids are substituted with any combination of one ormore conservative amino acid substitutions, non-conservative amino acidsubstitutions, non-naturally occurring amino acid substitutions,non-standard amino acids, and/or substitutions with amino acid analogsand further wherein said polypeptides are non-immunogenic or exhibitreduced immunogenicity as determined and assayed by comparison toimmunogenicity of corresponding non-amino acid substituted forms of PE;as measured using in vitro or in vivo assays. In particular embodiments,amino acid substituted forms of PE are at least 25%, at least about 25%,at least 50%, at least about 50%, at least 75%, or at least about 75%less immunogenic compared to corresponding non-amino acid substitutedforms of PE. In particular embodiments, amino acid substituted forms ofPE are at least 2-fold, at least about 2-fold, at least 3-fold, at leastabout 3-fold, at least 4-fold, at least about 4-fold, at least 5-fold,at least about 5-fold, at least 10-fold, at least about 10-fold, atleast 50-fold, at least about 50-fold, at least 100-fold, at least about100-fold, at least 500-fold, at least about 500-fold, at least1000-fold, or at least about 1000-fold less immunogenic compared tocorresponding non-amino acid substituted forms of PE. In one embodiment,amino acid substituted forms of PE are non-immunogenic or exhibitundetectable immunogenicity compared to corresponding non-amino acidsubstituted forms of PE.

The immunogenicity of substituted peptides may be measured via assaysroutinely known and used by those of skill in the art. For example,immunogenicity may be assayed by methods including, but not limited to,the proliferation assays described in Example 1 herein.

Additionally, methods for predicting, and assays for assessing,immunogenicity include those methods and assays such as described orreferenced in:

-   Baker M P and Jones T D. Identification and removal of    immunogenicity in therapeutic proteins. Curr. Opin. Drug. Disc. Dev.    2007 10(2): 219-227.-   Bryson C J, Jones T D, Baker M P. Prediction of immunogenicity of    therapeutic proteins: validity of computational tools. BioDrugs.    2010; 24(1):1-8.-   Chester, K, Baker, M P and Mayer A. Overcoming the immunologic    response to foreign enzymes in cancer therapy. Expert Rev. Clin.    Immunol. 2006 1(4): 549-559.-   Hochuli E. Interferon immunogenicity: technical evaluation of    interferon-alpha 2a. J Interferon Cytokine Res. 1997 17 Suppl    1:S15-21.-   Jaber A and Baker M P. Assessment of the immunogenicity of different    interferon beta-1a formulations using ex vivo T cell assays. J Pharm    Biomed Anal 2007 43(4):1256-61.-   Perry L C, Jones T D and Baker M P. New approaches to prediction of    immune responses to therapeutic proteins during preclinical    development. Drugs R D. 2008 9(6):385-96.-   Schellekens, H., Ryff, J. C., and Van Der Meide, P. H. Assays for    antibodies to human interferon-alpha: the need for    standardization. J. Interferon Cytokine Res. 1997 17(Suppl. 1),    S5-S8.

Embodiments of the invention include polypeptides comprising PE whereinone or more amino acids are substituted with any combination of one ormore conservative amino acid substitutions, non-conservative amino acidsubstitutions, non-naturally occurring amino acid substitutions,non-standard amino acids, and/or substitutions with amino acid analogsand further wherein said polypeptides retain biological activity asdetermined and assayed by comparison to biological activities ofcorresponding non-amino acid substituted forms of PE; such as, but notlimited to, cell killing activity, cell cytotoxicity, inactivation ofthe translation elongation factor EF-2, ADP-ribosylation of EF-2, andinhibition of protein synthesis as measured using in vitro or in vivoassays. In particular embodiments, amino acid substituted forms of PEexhibit 100% or about 100% of biological activity compared tocorresponding non-amino acid substituted forms of PE. In particularembodiments, amino acid substituted forms of PE exhibit at least 95%, orat least about 95% of biological activity compared to correspondingnon-amino acid substituted forms of PE. In particular embodiments, aminoacid substituted forms of PE exhibit at least 90%, at least about 90%,at least 85%, at least about 85%, at least 80%, at least about 80%, atleast 75%, at least about 75%, at least 70%, at least about 70%, atleast 60%, at least about 60%, at least 50%, or at least about 50% ofbiological activity compared to corresponding non-amino acid substitutedforms of PE.

Embodiments of the invention further comprise fusion proteins,conjugates, covalently-linked, and non-covalently linked amino acidsubstituted forms of PE, or fragments thereof, as described herein.Amino acid substituted forms of PE may be fused, conjugated or otherwiselinked with any artificial, recombinant, or naturally occurring moleculeor polypeptide to modify PE activity and/or PE localization/targeting,such as by conferring to PE, via said fusion or conjugation, the tissuetargeting, cell targeting, or sub-cellular localization properties ofthe molecule to which PE is fused, conjugated or otherwise linked. Forexample, but without limitation, amino acid substituted forms, orfragments thereof, of PE may be fused, conjugated, or otherwise linkedwith any type of antibody or antigen-binding fragments thereof,cell-surface receptor, secreted or cell-surface ligand, or fragmentsthereof.

In one embodiment, amino acid substituted forms of PE, including aminoacid substituted forms of PE fused, conjugated or otherwise linked toanother molecule or polypeptide are useful in the treatment of cancer;including, but not limited to, types of cancer described herein. In oneembodiment, amino acid substituted forms of PE as described herein areuseful for the preparation of a medicament for the treatment of cancer;including, but not limited to, types of cancer described herein.

In one embodiment, amino acid substituted forms of PE, or fragmentsthereof, may be fused, conjugated, or otherwise linked, withoutlimitation, antigen-binding moieties such as antibodies, or fragmentsthereof, which specifically or preferentially bind to disease associatedantigens. Such molecules include, for example, but without limitation,antibodies indicated in Table 1.

TABLE 1 Examples of Antibodies and Therapeutic Uses Putative Example(s)NAME TRADE NAME Antigen Targets of Therapeutic Use 3F8 GD2 neuroblastomaABAGOVOMAB CA-125 ovarian cancer (imitation) ABCIXIMAB REOPRO CD41(integrin platelet aggregation alpha-IIb) inhibitor ADALIMUMAB HUMIRATNF-α rheumatoid arthritis etc. ADECATUMUMAB EpCAM prostate and breastcancer AFELIMOMAB TNF-α sepsis AFUTUZUMAB CD20 lymphoma ALACIZUMABVEGFR2 cancer PEGOL ALD518 IL-6 rheumatoid arthritis ALEMTUZUMABCAMPATH, CD52 CLL, CTCL MABCAMPATH ALTUMOMAB HYBRI- CEA colorectalcancer PENTETATE CEAKER (diagnosis) ANATUMOMAB TAG-72 non-small celllung MAFENATOX carcinoma ANRUKINZUMAB IL-13 antigen-induced pulmonaryinflammation, asthma ATOLIZUMAB HLA-DR hematological cancers ARCITUMOMABCEA-SCAN CEA gastrointestinal cancers (diagnosis) ASELIZUMAB L-selectinseverely injured patients (CD62L) ATLIZUMAB ACTEMRA, IL-6 receptorrheumatoid arthritis ROACTEMRA ATOROLIMUMAB Rhesus factor hemolyticdisease of the newborn BAPINEUZUMAB beta amyloid Alzheimer's diseaseBASILIXIMAB SIMULECT CD25 (α chain of prevention of organ IL-2 receptor)transplant rejections BAVITUXIMAB phosphatidylserine cancer, viralinfections BECTUMOMAB LYMPHOSCAN CD22 non-Hodgkin's lymphoma (detection)BELIMUMAB BENLYSTA, BAFF non-Hodgkin lymphoma LYMPHOSTAT-B etc.BENRALIZUMAB CD125 asthma BERTILIMUMAB CCL11 (eotaxin-1) severe allergicdisorders BESILESOMAB SCINTIMUN CEA-related inflammatory lesions andantigen metastases (detection) BEVACIZUMAB AVASTIN VEGF-A metastaticcancer BICIROMAB FIBRISCINT fibrin II, beta chain thromboembolism(diagnosis) BIVATUZUMAB CD44 v6 squamous cell carcinoma MERTANSINEBLINATUMOMAB CD19 cancer BRENTUXIMAB CD30 (TNFRSF8) hematologic cancersVEDOTIN BRIAKINUMAB IL-12, IL-23 psoriasis, rheumatoid arthritis,inflammatory bowel diseases, multiple sclerosis CANAKINUMAB ILARIS IL-1rheumatoid arthritis CANTUZUMAB mucin CanAg colorectal cancer etc.MERTANSINE CAPROMAB PROSTASCINT prostatic prostate cancer (detection)PENDETIDE carcinoma cells CATUMAXOMAB REMOVAB EpCAM, CD3 ovarian cancer,malignant ascites, gastric cancer CC49 TAG-72 tumor detectionCEDELIZUMAB CD4 prevention of organ transplant rejections, treatment ofautoimmune diseases CERTOLIZUMAB CIMZIA TNF-α Crohn's disease PEGOLCETUXIMAB ERBITUX EGFR metastatic colorectal cancer and head and neckcancer CITATUZUMAB EpCAM ovarian cancer and other BOGATOX solid tumorsCIXUTUMUMAB IGF-1 receptor solid tumors CLENOLIXIMAB CD4 rheumatoidarthritis CLIVATUZUMAB MUC1 pancreatic cancer TETRAXETAN CONATUMUMABTRAIL-R2 cancer CR6261 Influenza A infectious disease/influenzahemagglutinin A DACETUZUMAB CD40 hematologic cancers DACLIZUMAB ZENAPAXCD25 (α chain of prevention of organ IL-2 receptor) transplantrejections DARATUMUMAB CD38 (cyclic ADP myleoma, CD3 8-positive ribosehydrolase) multiple myeloma DENOSUMAB PROLIA RANKL osteoporosis, bonemetastases etc. DETUMOMAB B-lymphoma cell lymphoma DORLIMOMAB autoimmune auto immune disorders ARITOX associated antigen DORLIXIMZUMAB CD3type 1 diabetes, autoimmune diseases ECROMEXIMAB GD3 gangliosidemalignant melanoma ECULIZUMAB SOLARIS C5 paroxysmal nocturnalhemoglobinuria EDOBACOMAB Endotoxin sepsis caused by Gram- negativebacteria EDRECOLOMAB PANOREX EpCAM colorectal carcinoma EFALIZUMABRAPTIVA LFA-1 (CD11a) psoriasis (blocks T-cell migration) EFUNGUMABMYCOGRAB Hsp90 invasive Candida infection ELOTUZUMAB SLAMF7 multiplemyeloma ELSILIMOMAB IL-6 Lymphoma, myeloma ENLIMOMAB/ ICAM-1 (CD54)stroke ENLIMOMAB PEGOL EPITUMOMAB Episialin cancer CITUXETAN EPRATUZUMABCD22 cancer, SLE ERLIZUMAB ITGB2 (CD18) heart attack, stroke, traumaticshock ERTUMAXOMAB REXOMUN HER2/neu, CD3 breast cancer etc. ETARACIZUMABABEGRIN integrin αvβ3 melanoma, prostate cancer, ovarian cancer etc.EXBIVIRUMAB hepatitis B surface hepatitis B antigen FANOLESOMABNEUTROSPEC CD15 appendicitis (diagnosis) FARALIMOMAB interferon receptorautoimmune disorders FARLETUZUMAB folate receptor 1 ovarian cancerFELVIZUMAB respiratory respiratory syncytial virus syncytial virusinfection FEZAKINUMAB IL-22 rheumatoid arthritis, psoriasis FIGITUMUMABIGF-1 receptor adrenocortical carcinoma, non-small cell lung carcinomaetc. FONTOLIZUMAB HUZAF IFN-γ Crohn's disease etc. FORAVIRUMAB rabiesvirus rabies (prophylaxis) glycoprotein FRESOLIMUMAB TGF-β idiopathicpulmonary fibrosis, focal segmental glomerulosclerosis, cancer GALIXIMABCD80 B-cell lymphoma GANTENERUMAB beta amyloid Alzheimer's diseaseGAVILIMOMAB CD147 (basigin) graft versus host disease GEMTUZUMABMYLOTARG CD33 acute myelogenous OZOGAMICIN leukemia GIRENTUXIMABRENCAREX carbonic clear cell renal cell anhydrase 9 carcinoma (CA-IX)GLEMBATUMUMAB GPNMB melanoma, breast cancer VEDOTIN GOLIMUMAB SIMPONITNF-α rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitisGOMILIXIMAB CD23 (IgE allergic asthma receptor) IBALIZUMAB CD4 HIVinfection IBRITUMOMAB ZEVALIN CD20 non-Hodgkin's lymphoma TIUXETANIGOVOMAB INDIMACIS-125 CA-125 ovarian cancer (diagnosis) IMCIROMABMYOSCINT cardiac myosin cardiac imaging INFLIXIMAB REMICADE TNF-αrheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis,psoriasis, Crohn's disease, ulcerative colitis INTETUMUMAB CD51 solidtumors (prostate cancer, melanoma) INOLIMOMAB CD25 (α chain of graftversus host disease IL-2 receptor) INOTUZUMAB CD22 cancer OZOGAMICINIPILIMUMAB YERVOY CD152 melanoma IRATUMUMAB CD30 (TNTRSF8) Hodgkin'slymphoma KELIXIMAB CD4 chronic asthma LABETUZUMAB CEA-CIDE CEAcolorectal cancer LEBRIKIZUMAB IL-13 asthma LEMALESOMAB NCA-90diagnostic agent (granulocyte antigen) LERDELIMUMAB TGF beta 2 reductionof scarring after glaucoma surgery LEXATUMUMAB TRAIL-R2 cancerLIBIVIRUMAB hepatitis B surface hepatitis B antigen LINTUZUMAB CD33cancer LORVOTUZUMAB CD56 cancer MERTANSINE LUCATUMUMAB CD40 multiplemyeloma, non- Hodgkin's lymphoma, Hodgkin's lymphoma LUMILIXIMAB CD23(IgE chronic lymphocytic receptor) leukemia MAPATUMUMAB TRAIL-R1 cancerMASLIMOMAB T-cell receptor autoimmune disorders MATUZUMAB EGFRcolorectal, lung and stomach cancer MEPOLIZUMAB BOSATRIA IL-5 asthma andwhite blood cell diseases METELIMUMAB TGF beta 1 systemic sclerodermaMILATUZUMAB CD74 multiple myeloma and other hematological malignanciesMINRETUMOMAB TAG-72 cancer MITUMOMAB GD3 ganglioside small cell lungcarcinoma MOROLIMUMAB Rhesus factor disease antigen MOTAVIZUMAB NUMAXrespiratory respiratory syncytial virus syncytial virus (prevention)MUROMONAB- ORTHOCLONE CD3 prevention of organ CD3 OKT3 transplantrejections NACOLOMAB C242 antigen colorectal cancer TAFENATOX NAPTUMOMAB5T4 non-small cell lung ESTAFENATOX carcinoma, renal cell carcinomaNATALIZUMAB TYSABRI integrin α4 multiple sclerosis, Crohn's diseaseNEBACUMAB Endotoxin sepsis NECITUMUMAB EGFR non-small cell lungcarcinoma NERELIMOMAB TNF-α auto immune disorders NIMOTUZUMAB THERACIM,EGFR squamous cell carcinoma, THERALOC head and neck cancer,nasopharyngeal cancer, glioma NOFETUMOMAB VERLUMA cancer-associatedcancer (diagnosis) MERPENTAN antigen OCRELIZUMAB CD20 rheumatoidarthritis, lupus erythematosus etc. ODULIMOMAB LFA-1 (CD11a) preventionof organ transplant rejections, immunological diseases OFATUMUMABARZERRA CD20 chronic lymphocytic leukemia OLARATUMAB PDGF-R α cancerOMALIZUMAB XOLAIR IgE Fc region allergic asthma OPORTUZUMAB EpCAM cancerMONATOX OREGOVOMAB OVAREX CA-125 ovarian cancer OTELIXIZUMAB CD3diabetes mellitus type 1 PAGIBAXIMAB lipoteichoic acid sepsis(Staphylococcus) PALIVIZUMAB SYNAGIS, respiratory respiratory syncytialvirus ABBOSYNAGIS syncytial virus (prevention) PANITUMUMAB VECTIBIX EGFRcolorectal cancer PANOBACUMAB Pseudomonas Pseudomonas aeruginosaaeruginosa infection PASCOLIZUMAB IL-4 asthma PEMTUMOMAB THERAGYN MUC1cancer PERTUZUMAB OMNITARG HER2/neu cancer PEXELIZUMAB C5 reduction ofside effects of cardiac surgery PINTUMOMAB adenocarcinoma adenocarcinoaantigen PRILIXIMAB CD4 Crohn's disease, multiple sclerosis PRITUMUMABvimentin brain cancer PRO 140 CCR5 HIV infection RAFIVIRUMAB rabiesvirus rabies (prophylaxis) glycoprotein RAMUCIRUMAB VEGFR2 solid tumorsRANIBIZUMAB LUCENTIS VEGF-A macular degeneration (wet form) RAXIBACUMABanthrax toxin, anthrax (prophylaxis and protective antigen treatment)REGAVIRUMAB cytomegalovirus cytomegalovirus infection glycoprotein BRESLIZUMAB IL-5 inflammations of the airways, skin and gastrointestinaltract RILOTUMUMAB HGF solid tumors RITUXIMAB MABTHERA, CD20 lymphomas,leukemias, RITUXAN some autoimmune disorders ROBATUMUMAB IGF-1 receptorcancer RONTALIZUMAB IFN-α systemic lupus erythematosus ROVELIZUMABLEUKARREST CD11, CD18 haemorrhagic shock RUPLIZUMAB ANTOVA CD154 (CD40L)rheumatic diseases SATUMOMAB TAG-72 cancer PENDETIDE SEVIRUMABcytomegalovinis cytomegalovirus infection SIBROTUZUMAB FAP cancerSIFALIMUMAB IFN-α SLE, dermatomyositis, polymyositis SILTUXIMAB IL-6cancer SIPLIZUMAB CD2 psoriasis, graft-versus-host disease (prevention)SOLANEZUMAB beta amyloid Alzheimer's disease SONEPCIZUMAB sphingosine-1-choroidal and retinal phosphate neovascularization SONTUZUMAB episialindisease antigen STAMULUMAB myostatin muscular dystrophy SULESOMALEUKOSCAN NCA-90 osteomyelitis (imaging) (granulocyte antigen)TACATUZUMAB AFP-CIDE alpha-fetoprotein cancer TETRAXETAN TADOCIZUMABintegtin αIIbβ3 percutaneous coronary intervention TALIZUMAB IgEallergic reaction TANEZUMAB NGF pain TAPLITUMOMAB CD19 cancer PAPTOXTEFIBAZUMAB AUREXIS clumping factor A Staphylococcus aureus infectionTELIMOMAB autoimmune autoimmune disorders ARITOX antigen TENATUMOMABtenascin C cancer TENELIXIMAB CD40 autoimmune disorders TEPLIZUMAB CD3diabetes mellitus type 1 TGN1412 CD2 chronic lymphocytic leukemia,rheumatoid arthritis TICILIMUMAB CTLA-4 cancer TIGATUZUMAB TRAIL-R2cancer TNX-650 IL-13 Hodgkin's lymphoma TOCILIZUMAB ACTEMRA, IL-6receptor rheumatoid arthritis ROACTEMRA TORALIZUMAB CD154 (CD40L)rheumatoid arthritis, lupus nephritis TOSITUMOMAB BEXXAR CD20 follicularlymphoma TRASTUZUMAB HERCEPTIN HER2/neu breast cancer TREMELIMUMABCTLA-4 cancer TUCOTUZUMAB EpCAM cancer CELMOLEUKIN TUVIRUMAB hepatitis Bvirus chronic hepatitis B URTOXAZUMAB Escherichia coli diarrhoea causedby E. coli USTEKINUMAB STELARA IL-12, IL-23 multiple sclerosis,psoriasis, psoriatic arthritis VAPALIXEMAB AOC3 (VAP-1) autoimmunedisorders VEDOLIZUMAB integtin α4β7 Crohn's disease, ulcerative colitisVELTUZUMAB CD20 non-Hodgkin's lymphoma VEPALEMOMAB AOC3 (VAP-1)inflammation VISILIZUMAB NUVION CD3 Crohn's disease, ulcerative colitisVOLOCIXIMAB integrin α5β1 solid tumors VOTUMUMAB HUMASPECT tumor antigencolorectal tumors CTAA16.88 ZALUTUMUMAB HUMAX- EGFR squamous cellcarcinoma of EGFR the head and neck ZANOLIMUMAB HUMAX-CD4 CD4 rheumatoidarthritis, psoriasis, T-cell lymphoma ZIRALIMUMAB CD147 (basigin)autoimmune disorders ZOLIMOMAB CD5 systemic lupus ARITOX erythematosus,graft-versus- host disease

In certain embodiments, amino acid substituted forms of PE, or fragmentsthereof, may be fused, conjugated, or otherwise linked, withoutlimitation, to naturally occurring normal or disease related moleculessuch as secreted, extracellular, intracellular, transmembrane, orcell-surface-bound molecules or fragments thereof (or non-naturallyoccurring variants and fragments thereof), such as without limitation:ligands, receptors, receptor extracellular domains, cytokines, growthfactors, cell signaling proteins, extracellular and intracellularenzymes, structural proteins, cell adhesion proteins and molecules,cluster of differentiation (CD) molecules, mitogens, cell divisionregulating molecules, cancer/tumor markers and antigens, et cetera. Incertain embodiments, molecules which are normally transmembrane andcell-surface bound polypeptides may be fused or conjugated to amino acidsubstituted forms of PE as polypeptide fragments lacking at least theirtransmembrane domains or polypeptide regions responsible forcell-surface binding.

In certain embodiments, molecules may be fused or conjugated to aminoacid substituted forms of PE wherein such molecules possess or retainthe ability (even as fusion proteins or protein conjugates) to formmultimeric complexes (such as hetero- and homopolymers including, butnot limited to, dimers, trimers, tetramers, pentamers, hexamers, etcetera.)

In certain embodiments, amino acid substituted forms of PE, or fragmentsthereof, may be generated as in-frame polypeptide fusion proteins withmolecules (such as, but not limited to, those referenced above) whereinthe PE moiety is either an amino-terminal portion or a carboxyl-terminalportion of the fusion protein. Determination of which of these twoconfigurations provides the desired results and/or biological activitiesmay be determined by routine experimentation practiced by those skilledin the art.

In certain embodiments, amino acid substituted forms of PE, or fragmentsthereof, may be generated as fusion proteins wherein heterologous aminoacid sequences (such as cell targeting sequences) are inserted withinthe amino acid substituted form of PE (i.e., heterologous amino acidsare flanked at the amino terminus and at the carboxy terminus by PEamino acid sequences). An example of a non-amino acid substituted formof PE in such a configuration is demonstrated in U.S. Pat. No. 8,854,044wherein a TGF-α polypeptide is incorporated at amino acid residues 607to 604 within a “PE37” polypeptide sequence. See e.g., U.S. Pat. No.8,854,044, FIG. 1.

Some examples of molecules which may be fused, conjugated, or otherwiselinked to amino acid substituted forms of PE, include for example, butwithout limitation, those such as indicated in Table 2.

TABLE 2 Examples of Potential Compounds and Indications to which Amino Acid Substituted Forms of PE MayBe Fused or Conjugated for Therapeutic Use Example Molecule (NucleotideAccession*) [Protein Potential Accession**] IndicationsExample Amino Acid Sequence Mesothelin Pancreatic cancerMALPTARPLLGSCGTPALGSLLFLLFSLGWVQPS (NM_013404) Ovarian cancerRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLG [NP_037536]FPCAEVSGLSTERVRELAVALAQKNVKLSTEQLR CLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACW GVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPS TWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGK KAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGY LFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGG APTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDD LDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 167) CD24 Liver cancerMGRAMVARLGLGLLLLALLLPTQIYSSETTTGTS (NM_013230) Colorectal cancerSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASL [AAH64619] Pancreatic cancerFVVSLSLLHLYS (SEQ ID NO: 168) CD22 Hairy CellVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEM (AB013007) LeukemiaNIPRTG (SEQ ID NO: 169) [BAA36576] Chronic Lymphocytic LeukemiaNon-Hodgkin's Lymphoma CD25 Hodgkin's MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHAa.k.a., Interleukin Lymphoma TFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCT2 receptor, alpha Hairy Cell GNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQK chainLeukemia ERKTTEMQSPMQPVDQASLPGHCREPPPWENEAT (NM_000417) ChronicERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKM [NP_000408] LymphocyticTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPE LeukemiaGRPESETSCLVTTTDFQIQTEMAATMETSIFTTE Cutaneous T-cellYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI Lymphoma (SEQ ID NO: 170)Adult T-cell leukemia CD174 Bladder cancerMDPLGAAKPQWPWRRCLAALLFQLLVAVCFFSYL a.k.a., Lewis Y, Breast cancerRVSRDDATGSPRAPSGSSRQDTTPTRPTLLILLW galactoside 3(4)- Colorectal cancerTWPFHIPVALSRCSEMVPGTADCHITADRKVYPQ L- Esophageal cancerADTVIVHHWDIMSNPKSRLPPSPRPQGQRWIWFN fucosyltransferase Gastric cancerLEPPPNCQHLEALDRYFNLTMSYRSDSDIFTPYG (NM_000149) Lung cancerWLEPWSGQPAHPPLNLSAKTELVAWAVSNWKPDS [NP_000140] Pancreatic cancerARVRYYQSLQAHLKVDVYGRSHKPLPKGTMMETL SRYKFYLAFENSLHPDYITEKLWRNALEAWAVPVVLGPSRSNYERFLPPDAFIHVDDFQSPKDLARYL QELDKDHARYLSYFRWRETLRPRSFSWALDFCKACWKLQQESRYQTVRSIAAWFT (SEQ ID NO: 171) TPBG Non small cell MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSP a.k.a., oncofetal lung cancerTSSASSFSSSAPFLASAVSAQPPLPDQCPALCEC antigen 5T4, 5T4 Renal carcinomaSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQ oncofetal Pancreatic cancerLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAG trophoblastAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVS glycoproteinAPSPLVELILNHIVPPEDERQNRSFEGMVVAALL (NM_006670)AGRALQGLRRLELASNHFLYLPRDVLAQLPSLRH [CAA09930]LDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKV LHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSA DLDCDPILPPSLQTSYVFLGIVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYHYRYEINAD PRLTNLSSNSDV (SEQ ID NO: 172) CD56Small cell lung MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVG a.k.a., NCAM1, cancerESKFFLCQVAGDAKDKDISWFSPNGEKLTPNQQR neural cell Merkel cellISVVWNDDSSSTLTIYNANIDDAGIYKCVVTGED adhesion carcinomaGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAV molecule 1 Ovarian cancerIVCDVVSSLPPTIIWKHKGRDVILKKDVRFIVLS isoform 1 NeuroendocrineNNYLQIRGIKKTDEGTYRCEGRILARGEINFKDI precursor tumorsQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAE (NM_000615) Multiple MyelomaGFPEPTMSWTKDGEQIEQEEDDEKYIFSDDSSQL [NP_000606]TIKKVDKNDEAEYICIAENKAGEQDATIHLKVFA KPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRTSTRNISSEEKTLDGHMVVRSHARVSSLTLKS IQYTDAGEYICTASNTIGQDSQSMYLEVQYAPKLQGPVAVYTWEGNQVNITCEVFAYPSATISWFRDG QLLPSSNYSNIKIYNTPSASYLEVTPDSENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEP YSSTAQVQFDEPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPETTYAVRLAA LNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKVNLIKQDDGGSPIRHYLVRYRALSSEW KPEIRLPSGSDHVMLKSLDWNAEYEVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAI VGILIVIFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFSKDESKEPIVEVR TEEERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQETETKPAPAEVKTVPNDATQTKENESKA (SEQ ID NO: 173) C-type lectin-Acute myeloid MWIDFFTYSSMSEEVTYADLQFQNSSEMEKIPEI like molecule-1leukemia GKFGEKAPPAPSHVWRPAALFLTLLCLLLLIGLG a.k.a., CLL-1VLASMFHVTLKIEMKKMNKLQNISEELQRNISLQ (AY547296)LMSNMNISNKIRNLSTTLQTIATKLCRELYSKEQ [AAT11783]EHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMA CAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCG YINRLYVQYYHCTYKQRMICEKMANPVQLGSTYFREA (SEQ ID NO: 174) *“Nucleotide Accession” refers to the NCBIReference Sequence accession number associated with the correspondingnucleic acid sequence as found in the “Nucleotide” database provided forpublic access and searching (via the Internet) through the NationalCenter for Biotechnology Information, U.S. National Library of Medicine(8600 Rockville Pike, Bethesda MD, 20894 USA (www.ncbi.nlm.nih.gov)).**“Protein Accession” refers to the NCBI Reference Sequence accessionnumber associated with the corresponding amino acid sequence as found inthe “Protein” database provided for public access and searching (via theInternet) through the National Center for Biotechnology Information,U.S. National Library of Medicine (8600 Rockville Pike, Bethesda MD,20894 USA (www.ncbi.nlm.nih,gov)).Note: The potential indications and nucleic acid and amino acidsequences shown in Table 2 (as well as accession numbers listed) arepresented for purposes of providing a few illustrative examples only.Thus, embodiments of the invention may or may not comprise theseindications and sequences. Accordingly, it is envisioned thatembodiments of the invention comprise other indication uses as well asother molecules and sequence variants (e.g., naturally occurringvariants (such as allelic or polymorphic variants) and non-naturallyoccurring variants (such as genetically engineered or mutated variants))of these sequences wherein one or multiple amino acids are changedand/or wherein only a fragment or fragments of such sequences are fusedor conjugated to amino acid substituted forms of PE. Hence, the examplesshown in Table 2 should in no manner be considered limiting with respectto potential therapeutic indications or protein fusions and conjugatesof amino acid modified forms of PE.

In one embodiment, the present invention includes isolated nucleic acidsand methods of expressing nucleic acids encoding any of theherein-referenced modified forms of PE, including fusions, conjugates,and otherwise linked molecules; whether such forms are expressed from asingle or one more separate polynucleotide sequences; whether suchpolynucleotide sequences are expressed from a single or one or moreseparate expression vectors.

Expression Vectors

In one embodiment, the present invention includes methods of making andusing recombinant expression vectors to express nucleic acids encodingpolypeptides comprising any of the herein-referenced modified forms ofPE, including fusions, conjugates, and otherwise linked molecules. Useof a wide variety of expression vectors are well-known and routinelyused by those skilled in the art. A few examples of the types ofexpression vectors which may be used include, but are not limited to:derivatives of human or animal viruses (such as retrovirus,adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex,Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors) andinsect viruses (such as baculovirus); yeast vectors; bacteriophagevectors (e.g., bacteriophage lambda); plasmids; cosmids; artificialchromosomes; liposomes; electrically charged lipids (cytofectins);DNA-protein complexes, and biopolymers.

Gene Delivery and Expression Systems

A wide variety of methods (i.e., gene delivery systems) are availableand well-known to those of skill in the art; any of such methods may beused for introducing nucleic acids encoding modified forms of PE into acell, tissue, or organism for in vitro, in vivo, in situ, or ex vivoexpression. The methods referenced below represent examples of ways inwhich nucleic acid(s) encoding modified forms of PE may be introducedinto a cell. These examples are in no way intended to limit the scope ofthat may be used for gene delivery and expression of modified forms ofPE in cells, tissues, or organisms; these examples are presented toillustrate the many available methods.

—Viral-Based Delivery of Target Nucleic Acids—

Gene therapy based methods can be used to deliver (into a host cell,tissue or organism) target nucleic acids encoding modified forms of PE(and other polynucleotides, as needed, to allow expression of the same).As one example, polynucleotides operably encoding the target nucleicacid can be delivered to a tissue or organism either as “naked nucleicacid” or as part of an expression vector. The term vector includes forexample, but is not limited to, vectors such as plasmid vectors, cosmidvectors, artificial chromosome vectors, and viral vectors. Some examplesof viral vectors include adenovirus, herpes simplex virus (HSV),alphavirus, simian virus 40, picomavirus, vaccinia virus, retrovirus,lentivirus, and adeno-associated virus. Vectors encoding modified formsof PE may be capable of replication in a cell in which it is introduced,or it may be preferred that the vector is not capable of replication.Vectors encoding modified forms of PE may be capable of integration intothe genomic DNA of a cell (and subsequent expression therefrom), or itmay be preferred that the vector is not capable of integrating into thehost genome. An example of a vector that can integrate into the genomicDNA of a cell is a retroviral vector, in which an integrase enzymemediates integration of the retroviral vector sequences. A vector mayalso contain transposon sequences that facilitate integration of thecoding region into the genomic DNA of a host cell. Liposomes representanother manner in which target DNA may be delivered to a subject.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, type of target host cell, species of host organism,desired duration of protein expression. An expression vector optionallyincludes expression control sequences operably linked to the codingsequence such that the coding region is expressed in the cell. Theinvention is not limited by the use of any particular promoter, and awide variety is known. Promoters act as regulatory signals that bind RNApolymerase in a cell to initiate transcription of a downstream (3′direction) operably linked coding sequence. The promoter used in theinvention may be a constitutive or an inducible promoter. It can be, butneed not be, heterologous with respect to the cell to which it isintroduced.

In certain embodiments, adenovirus expression vectors can be used todeliver (into a host cell, tissue or organism) target nucleic acidsencoding modified forms of PE (and other polynucleotides, as needed, toallow expression of the same). The terms “adenovirus expression vector”is meant to include those constructs containing nucleic acid sequencessufficient to (a) support packaging of the construct and (b) toultimately express a recombinant gene construct that has been insertedtherein. In contrast to retroviruses, use of adenovirus vectors does notresult in chromosomal integration because adenovirus DNA replicates inan episomal manner. Moreover, adenoviruses are considered to bestructurally stable with no genome rearrangement occurring even afterextensive virus reproduction and amplification. Methods of constructingand using adenovirus vectors as gene delivery systems are well-known tothose of skill in the art.

In certain embodiments, adeno-associated virus (AAV) expression vectorscan be used to deliver (into a host cell, tissue or organism) targetnucleic acids encoding modified forms of PE (and other polynucleotides,as needed, to allow expression of the same). AAV may be desirable for anumber or reasons; for example, because AAV vectors exhibit a highfrequency of integration, can infect nondividing cells, and have a broadhost range. AAV is a dependent parvovirus in that it requirescoinfection with another virus (either adenovirus or a member of theherpes virus family) to undergo a productive infection in culturedcells. In the absence of coinfection with helper virus, the wild-typeAAV genome integrates through its ends into a human chromosome where itresides as a latent provirus. When a cell containing latent AAV provirusis superinfected with a helper virus, the AAV genome is “rescued” fromthe chromosome and a normal productive infection is established. Methodsof constructing and using AAV vectors as gene delivery systems arewell-known to those of skill in the art.

In certain embodiments, retrovirus expression vectors can be used todeliver (into a host cell, tissue or organism) target nucleic acidsencoding modified forms of PE (and other polynucleotides, as needed, toallow expression of the same). Retroviruses are a group ofsingle-stranded RNA viruses characterized by the ability to converttheir genomic RNA to double-stranded DNA in infected cells through areverse-transcription process. The resulting DNA stably integrates intocellular chromosomes as a provirus and directs synthesis of viralproteins. Retroviral integration results in the retention of viral genesequences in the recipient cell and in its descendants. Retroviralvectors are able to infect a broad variety of cell types. Methods ofconstructing and using retroviruses as gene delivery systems arewell-known to those of skill in the art.

Many other expression vectors can also be used to deliver (into a hostcell, tissue or organism) target nucleic acids encoding modified formsof PE (and other polynucleotides, as needed, to allow expression of thesame). For example, vectors derived from viruses such as vacciniaviruses, herpes viruses, equine encephalitis viruses, hepatitis virusesand lentiviruses can be used. Methods of constructing and using viralexpression vectors as gene delivery systems are well-known to those ofskill in the art. The examples of such vectors referenced herein are notintended to be limiting with respect to the means by which modifiedforms of PE may be delivered and expressed in various host cells,tissues, or organisms.

—Non-Viral Delivery of Modified Target Nucleic Acids—

In addition to viral delivery of modified target nucleic acid, thefollowing are additional methods of recombinant gene delivery can beused to deliver (into a host cell, tissue or organism) target nucleicacids encoding modified forms of PE (and other polynucleotides, asneeded, to allow expression of the same). Methods of constructing andusing non-viral gene delivery systems are well-known to those of skillin the art. See, for example, Al-Dosari et al., “Nonviral gene delivery:principle, limitations, and recent progress,” AAPS Journal,11(4):671-681 (2009); and references cited therein.

In certain embodiments, electroporation can be used to deliver (into ahost cell, tissue or organism) target nucleic acids encoding modifiedforms of PE (and other polynucleotides, as needed, to allow expressionof the same). Methods of using electroporation are well-known to thoseof skill in the art. See, for example, Bodles-Brakhop et al.,“Electroporation for the delivery of DNA-based vaccines andimmunotherapeutics: current clinical developments,” Mol. Ther.,17(4):585-592 (2009); and references cited therein. See also, Golzio etal., “Observations of the mechanisms of electromediated DNA uptake-fromvesicles to tissues,” Curr Gene Ther., 10(4):256-266 (2010); andreferences cited therein. See also, Andre et al., “Nucleic acidselectrotransfer in vivo: mechanisms and practical aspects,” Curr GeneTher., 10(4):267-280 (2010); and references cited therein. See also,Wells, “Electroporation and ultrasound enhanced non-viral gene deliveryin vitro and in vivo,” Cell Biol Toxicol., 26(1):21-28 (2010); andreferences cited therein.

In certain embodiments, particle bombardment can be used to deliver(into a host cell, tissue or organism) target nucleic acids encodingmodified forms of PE (and other polynucleotides, as needed, to allowexpression of the same). This method depends on the ability toaccelerate nucleic acid-coated microprojectiles to a sufficient velocityto allow them to pierce cell membranes, thereby delivering nucleic acid“payloads,” without killing them. Some typical microprojectiles consistof biologically inert substances such as tungsten, platinum, and goldbeads. Methods of using particle bombardment are well-known to those ofskill in the art. See, for example, Klein et al., “Particle bombardment:a universal approach for gene transfer to cells and tissues,” Curr.Opin. Biotechnol., 4(5):583-590 (1993); and references cited therein.

In certain embodiments, a variety of methods incorporating calciumphosphate co-precipitation can be used to deliver (into a host cell,tissue or organism) target nucleic acids encoding modified forms of PE(and other polynucleotides, as needed, to allow expression of the same).Methods of using calcium phosphate co-precipitation are well-known tothose of skill in the art. See, for example, Uskoković et al.,“Nanosized hydroxyapatite and other calcium phosphates: chemistry offormation and application as drug and gene delivery agents,” J. Biomed.Mater. Res. B Appl. Biomater, 96(1):152-191 (2011); and references citedtherein. See also, Colosimo et al., “Transfer and expression of foreigngenes in mammalian cells,” Biotechniques, 29(2):314-8, 320-322 (2000);and references cited therein.

In certain embodiments, microinjection and sonication methods can beused to deliver (into a host cell, tissue or organism) target nucleicacids encoding modified forms of PE (and other polynucleotides, asneeded, to allow expression of the same). Methods of usingmicroinjection and sonication are well-known to those of skill in theart. See, for example, Rochlitz et al., “Gene therapy of cancer,” SwissMed. Wkly., 131(1-2):4-9 (2001); and references cited therein. See also,Donnelly et al., “Microneedle-based drug delivery systems:microfabrication, drug delivery, and safety,” Drug Deliv., 17(4):187-207 (2010); and references cited therein. See also, Miller et al.,“Sonoporation: mechanical DNA delivery by ultrasonic cavitation”, Somat.Cell Mol. Genet., 27(1-6): 115-34 (2002); and references cited therein.

In certain embodiments, liposomes and lipid formulations can be used todeliver (into a host cell, tissue or organism) target nucleic acidsencoding modified forms of PE (and other polynucleotides, as needed, toallow expression of the same). Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. An example of a commonly used,commercially available lipid formulation is Lipofectamine (Gibco BRL).Methods of using liposomes and lipid formulations to deliver nucleicacids to cells, tissues and organisms are well-known to those of skillin the art. See, for example, Xiong et al., “Cationic liposomes as genedelivery system: transfection efficiency and new application,”Pharmazie, 66(3):158-64 (2011); and references cited therein. See also,Pichon et al., “Chemical vectors for gene delivery: uptake andintracellular trafficking,” Curr Opin Biotechnol., 21(5):640-645 (2010);and references cited therein. See also, Pathak et al., “Recent trends innon-viral vector-mediated gene delivery,” Biotechnol J., 4(11):1559-1572 (2009).

Expression of Modified Forms of PE Via Gene Switch Modulation Systems

Expression of modified forms of PE, including fusions, conjugates, andotherwise linked molecules, may be expressed in host cells, tissues, andorganisms using gene switch expression systems. Some examples, withoutlimitation, of such gene expression systems, and genetically engineeredcells comprising gene switch expression systems, which can be used toexpress polynucleotides and polypeptides of the present invention, aredescribed in the following publications; each of which are herebyincorporated by reference herein:

WO 2009/045370 (PCT/US2008/011270); WO 2009/025866 (PCT/US2008/010040);WO 2002/066614 (PCT/US/2002/005706); WO 2008/073154 (PCT/US2007/016747);WO 2002/066613 (PCT/US2002/005090); WO 2005/108617 (PCT/US2005/015089);WO 2002/029075 (PCT/US2001/030608); WO 2003/0/27289 (PCT/US2002/005026);WO 2001/070816 (PCT/US2001/090500); WO 2002/066615 (PCT/US2002/005708);WO 2009/048560 (PCT/US2008/011563); WO 2003/027266 (PCT/US/2002/05234);WO 2010/042189 (PCT/US2009/005510); and WO 2002/066612(PCT/US2002/005090); WO 2011/119773 (PCT/US2011/029682).

For purposes of expressing polynucleotides and polypeptides undercontrol of a gene switch mechanism, the term “gene switch” refers to thecombination of a response element associated with a promoter, and aligand-dependent transcription factor-based system which, in thepresence of one or more ligands, modulates the expression of a gene intowhich the response element and promoter are incorporated. Statedotherwise, a “gene switch” refers to a peptide, protein or polypeptidecomplex that functions to (a) bind an activating ligand, and (b)regulate the transcription of a gene of interest in a ligand-dependentfashion.

In one embodiment, the polynucleotide encoding a gene switch is arecombinant polynucleotide, i.e., a polynucleotide, that has beenengineered, by molecular biological manipulation, to encode the geneswitch. In another embodiment, the recombinant polynucleotide is asynthetic polynucleotide.

As used herein with respect to gene switch regulation systems, the term“dimerizes with the ligand binding domain that binds an activatingligand” refers to a selective protein-protein interaction that isinduced by the presence of activating ligand.

As used herein, the term “ligand binding domain that binds an activatingligand” refers to an amino acid sequence that selectively binds anactivating ligand. In the methods disclosed herein, an activating ligandbinds to a ligand binding domain, e.g., an ecdysone receptor ligandbinding domain, that is part of a ligand-dependent transcriptionalactivation complex that regulates the expression of a polynucleotidesequence that encodes a gene of interest. Hence, the expression of thegene of interest is regulated in a ligand-dependent fashion.

The term “ecdysone receptor-based,” with respect to a gene switch,refers to a gene switch comprising at least a functional part of anaturally occurring or synthetic ecdysone receptor ligand binding domainand which regulates gene expression in response to a ligand that bindsto the ecdysone receptor ligand binding domain.

As used herein, “selective binding” of an activating ligand to a ligandbinding domain in a gene switch means that the ligand has an EC50 ofabout 700 nanomolar (nM), 650 nM, 600 nM, 550 nM, 500 nM, 450 nM, 400nM, 350 nM, 300 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM 70 nM, 65 nM, 60 nM, 55 nM, 50 nM,45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM or 1 nM, or less, in a gene switchassay.

As used herein, “EC50” is the “half maximal effective concentration,”which refers to the concentration of an activating ligand that induces agene switch-regulated change in expression of a polynucleotide encodingan gene of interest (e.g., modified forms of PE, including fusions,conjugates, et cetera), that is halfway between the baseline level ofexpression and the maximum level of expression after a specifiedexposure time. Examples of cellular assays for measuring geneswitch-regulated gene expression are well known to those of skill in theart. See, for example, Karzenowski et al., BioTechniques 39: 191-200(2005).

In one embodiment, the ligand binding domain that binds an activatingligand, e.g., an ecdysone receptor ligand binding domain, dimerizes withanother ligand binding domain, e.g., a retinoid X receptor ligandbinding domain, to form a protein-protein complex.

In one embodiment, the expression of the gene of interest is regulatedby an activating ligand in an on/off fashion that is independent of theconcentration or dosage of an activating ligand. In another embodiment,the expression of the gene of interest is regulated by an activatingligand in a concentration (or dosage)-dependent fashion, i.e., there isa dose-response relationship between the concentration (or dosage) of anactivating ligand and the level of gene expression of the gene ofinterest. See, e.g., US Patent Publication No. 2009/0123441 (see also,WO 2009/048560 (PCT/USUS2008/011563)).

The term “operably linked” refers to the association of polynucleotidesequences on a single polynucleotide so that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence when it is capable of affecting the expression of thatcoding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

In one embodiment, an activating ligand, or a composition thereof, isadministered to a subject orally. In another embodiment, an activatingligand, or a composition thereof, is administered to a subjectparenterally. In another embodiment, an activating ligand, or acomposition thereof, is administered subcutaneously, intramuscularly,intravenously, intraperitoneally, transdermally, or intratumorally.

In one embodiment, the ligand binding domain in the gene switch is aGroup H nuclear receptor ligand binding domain, or a mutant thereof thatbinds an activating ligand. In another embodiment, the Group H nuclearreceptor ligand binding domain is selected from the group consisting ofan ecdysone receptor ligand binding domain, a ubiquitous receptor ligandbinding domain, an orphan receptor-1 ligand binding domain, an NER-1ligand binding domain, a receptor-interacting protein-15 ligand bindingdomain, a liver X receptor-3 ligand binding domain, a steroid hormonereceptor-like protein ligand binding domain, a liver X receptor ligandbinding domain, a liver X receptor ligand binding domain, a farnesoid Xreceptor ligand binding domain, a receptor-interacting protein-14 ligandbinding domain, and a farnesol receptor ligand binding domain ligandbinding domain, or a mutant thereof that binds an activating ligand.

In another embodiment, the Group H nuclear receptor ligand bindingdomain is an ecdysone receptor ligand binding domain, or a mutantthereof that binds an activating ligand. In another embodiment, theecdysone receptor ligand binding domain is selected from the groupconsisting of an Arthropod ecdysone receptor ligand binding domain aLepidopteran ecdysone receptor ligand binding domain, a Dipteranecdysone receptor ligand binding domain, an Orthopteran ecdysonereceptor ligand binding domain, a Homopteran ecdysone receptor ligandbinding domain and a Hemipleran ecdysone receptor ligand binding domain,a spruce budworm Choristoneura fumiferana ecdysone receptor ligandbinding domain, a beetle Tenebrio molitor ecdysone receptor ligandbinding domain, a Manduca sexta ecdysone receptor ligand binding domain,a Heliothies virescens ecdysone receptor ligand binding domain, a midgeChironomus tentans ecdysone receptor ligand binding domain, a silk mothBombyx mori ecdysone receptor ligand binding domain, a squinting bushbrown Bicyclus anynana ecdysone receptor ligand binding domain, abuckeye Junonia coenia ecdysone receptor ligand binding domain, a fruitfly Drosophila melanogaster ecdysone receptor ligand binding domain, amosquito Aedes aegpti ecdysone receptor ligand binding domain, a blowflyLucilia capitata ecdysone receptor ligand binding domain, a blowflyLucilia cuprina ecdysone receptor ligand binding domain, a blowflyCalliphora vicinia ecdysone receptor ligand binding domain, aMediterranean fruit fly Ceratitis capitata ecdysone receptor ligandbinding domain, a locust Locusta migratoria ecdysone receptor ligandbinding domain, an aphid Myzus persicae ecdysone receptor ligand bindingdomain, a fiddler crab Celuca pugilator ecdysone receptor ligand bindingdomain, an ixodid tick Amblyomma americanum ecdysone receptor ligandbinding domain, a whitefly Bamecia argentifoli ecdysone receptor ligandbinding domain, a leafhopper Nephotetix cincticeps ecdysone receptorligand binding domain, or a mutant thereof that binds An activatingligand.

In another embodiment, the ecdysone receptor ligand binding domain is aspruce budworm Choristoneura fumiferana ecdysone receptor ligand bindingdomain, for which the amino acid sequence is:

(SEQ ID NO: 1) Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala Arg LeuIle Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser AspGlu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln GlnAla Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro PheArg Gln Ile Thr Glu Met Thr Ile Leu Thr Val GlnLeu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly PheAla Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu LeuLys Ala Cys Ser Ser Glu Val Met Met Leu Arg ValAla Arg Arg Tyr Asp Ala Ala Ser Asp Ser Val(position 107) Leu Phe Ala Asn Asn Gln Ala TyrThr Arg Asp Asn Tyr Arg Lys Ala Gly Met ala Tyr(position 127) Val Ile Glu Asp Leu Leu His PheCys Arg Cys Met Tyr Ser Met ala Leu Asp Asn IleHis Tyr Ala Leu Leu Thr Ala Val Val Ile Phe SerAsp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val GluGlu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg IleTyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg SerSer Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu SerGlu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn MetCys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu ProPro Phe Leu Glu Glu Ile Trp Asp Val,which is also set forth as SEQ NO: 1 in U.S. Patent Publication No.2006/0100416 A1 (see also, WO 2002/066612 (PCT/US2002/005090)).

Exemplary ecdysone receptor ligand binding domains include thosedisclosed, for example, in U.S. Pat. No. 7,935,510 (see also, WO2003/0/27289 (PCT/US2002/005026)); U.S. Pat. No. 7,919,269 (see also, WO2003/027266 (PCT/US/2002/05234)); U.S. Pat. No. 7,563,879 (see also, WO2003/0/27289 (PCT/US2002/005026)); and in U.S. Patent Publication No.2006/0100416 A1 (see also, WO 2002/066612 (PCT/US2002/005090)), each ofwhich is hereby incorporated by reference in its entirety.

In one embodiment, the ecdysone receptor ligand binding domain is amutant of an ecdysone receptor ligand binding domain that binds theactivating compound. In another embodiment, the ecdysone receptor ligandbinding domain is a mutant of the spruce budworm Choristoneurafumiferana ecdysone receptor ligand binding domain that binds theactivating compound.

In one embodiment, the gene switch comprises a Choristoneura fumiferanaecdysone receptor ligand binding domain that is engineered to containthe mutations V107I and Y127E of the Choristoneura fumiferana ecdysonereceptor sequence as set forth in SEQ ID NO: 1 of U.S. PatentPublication No. 2006/0100416 (see also, WO 2002/066612(PCT/US2002/005090)). The term “V107I” means that the valine amino acidresidue at position 107 (a as set forth in SEQ ID NO:1 of U.S. PatentPublication No. 2006/0100416) is changed to isoleucine. The term “Y127E”means that the tyrosine amino acid residue at position 127 (as set forthin SEQ ID NO:1 of U.S. Patent Publication No. 2006/0100416) is changedto glutamate.

Exemplary mutant ecdysone receptor ligand binding domains are disclosed,for example, in US 2006/0100416 A1 (see also, WO 2002/066612(PCT/US2002/005090)) and U.S. Pat. No. 7,935,510 (Pub. No. 2005/0266457)(see also, WO 2005/108617 (PCT/US2005/015089)) each of which isincorporated by reference in its entirety.

In one embodiment, the gene switch comprises a ligand binding domainthat dimerizes with the ligand binding domain that binds an activatingligand. In one embodiment, the ligand binding domain that dimerizes withthe ligand binding domain that binds an activating ligand is a Group Bnuclear receptor ligand binding domain. In another embodiment, the GroupB nuclear receptor ligand binding domain is selected from the groupconsisting of a retinoid X receptor ligand binding domain, an H-2 regionII binding protein ligand binding domain, a nuclear receptorco-regulator-1 ligand binding domain, an ultraspiracle protein ligandbinding domain, a 2C1 nuclear receptor ligand binding domain, and achorion factor 1 ligand binding domain. In another embodiment, a ligandbinding domain that dimerizes with the ligand binding domain that bindsan activating ligand is not an ecdysone receptor ligand binding domain.

In one embodiment, the ligand binding domain that dimerizes with theligand binding domain that binds an activating ligand is a retinoic Xreceptor ligand binding domain. In another embodiment, the retinoic Xreceptor ligand binding domain is a vertebrate retinoic X receptorligand binding domain. In another embodiment, the retinoic X receptorligand binding domain is a Homo sapiens retinoic X receptor ligandbinding domain. In another embodiment, the retinoic X receptor ligandbinding domain is a retinoic X receptor α isoform. In anotherembodiment, the retinoic X receptor ligand binding domain is a retinoicX receptor β isoform. In another embodiment, the retinoic X receptorligand binding domain is a retinoic X receptor γ isoform.

In another embodiment, the retinoic X receptor ligand binding domain isan invertebrate retinoic X receptor ligand binding domain. In anotherembodiment, the invertebrate retinoic X receptor ligand binding domainis a Locusta migratoria retinoic X receptor ligand binding domain.

In another embodiment, the invertebrate retinoic X receptor ligandbinding domain is a non-Lepidopteran, non-Dipteran retinoic X receptorligand binding domain.

In one embodiment, the retinoid receptor ligand binding domain is avertebrate retinoid X receptor ligand binding domain, an invertebrateretinoid X receptor ligand binding domain, an ultraspiracle proteinligand binding domain, or a chimeric retinoid X receptor ligand bindingdomain.

In one embodiment, the chimeric retinoid X receptor ligand bindingdomain comprises two polypeptide fragments, wherein the firstpolypeptide fragment is from a vertebrate retinoid X receptor ligandbinding domain, an invertebrate retinoid X receptor ligand bindingdomain, or an ultraspiracle protein ligand binding domain, and thesecond polypeptide fragment is from a different vertebrate retinoid Xreceptor ligand binding domain, a different invertebrate retinoid Xreceptor ligand binding domain, or a different ultraspiracle proteinligand binding domain.

In another embodiment, the chimeric retinoid X receptor ligand bindingdomain is one that is disclosed in U.S. Pat. No. 7,531,326, which ishereby incorporated by reference in its entirety.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-6, helices1-7, helices 1-8, helices 1-9, helices 1-10, helices 1-11, or helices1-12 of a first species of retinoid X receptor, and the secondpolypeptide fragment of the chimeric retinoid X receptor ligand bindingdomain comprises helices 7-12, helices 8-12, helices 9-12, helices10-12, helices 11-12, helix 12, or F domain of a second species ofretinoid X receptor, respectively.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-6 of afirst species RXR according to the disclosure, and the secondpolypeptide fragment of the chimeric retinoid X receptor ligand bindingdomain comprises helices 7-12 of a second species of retinoid Xreceptor.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-7 of afirst species retinoid X receptor according to the disclosure, and thesecond polypeptide fragment of the chimeric retinoid X receptor ligandbinding domain comprises helices 8-12 of a second species retinoid Xreceptor.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-8 of afirst species of retinoid X receptor, and the second polypeptidefragment of the chimeric retinoid X receptor ligand binding domaincomprises helices 9-12 of a second species of retinoid X receptor.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-9 of afirst species of retinoid X receptor, and the second polypeptidefragment of the chimeric retinoid X receptor ligand binding domaincomprises helices 10-12 of a second species of retinoid X receptor.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-10 of afirst species of retinoid X receptor, and the second polypeptidefragment of the chimeric retinoid X receptor ligand binding domaincomprises helices 11-12 of a second species of retinoid X receptor.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-11 of afirst species of retinoid X receptor, and the second polypeptidefragment of the chimeric retinoid X receptor ligand binding domaincomprises helix 12 of a second species of retinoid X receptor.

In another preferred embodiment, the first polypeptide fragment of thechimeric retinoid X receptor ligand binding domain comprises helices1-12 of a first species of retinoid X receptor, and the secondpolypeptide fragment of the chimeric retinoid X receptor ligand bindingdomain comprises an F domain of a second species of retinoid X receptor.

In one embodiment, the first polypeptide fragment in the chimericretinoid X receptor ligand binding domain is human retinoid X receptorsequence, and the second polypeptide fragment in the chimeric retinoid Xreceptor ligand binding domain is invertebrate retinoid X receptorsequence. In another embodiment, the invertebrate retinoid X receptorsequence is Locusta migratoria retinoid X receptor sequence.

In another embodiment, the first polypeptide fragment of the chimericretinoid X receptor ligand binding domain comprises helices 1-8 of ahuman retinoid X receptor, and the second polypeptide fragment of thechimeric retinoid X receptor ligand binding domain comprises helices9-12 of Locusta migratoria retinoid X receptor.

In one embodiment, the gene switch further comprises a DNA bindingdomain (“DBD”). In another embodiment, the DBD is selected from thegroup consisting of a GAL4 DBD, a LexA DBD, a transcription factor DBD,a steroid/thyroid hormone nuclear receptor superfamily member DBD, abacterial LacZ DBD, and a yeast DBD.

In one embodiment, the gene switch further comprises a transactivationdomain (“TD”). In another embodiment, the transactivation domain isselected from the group consisting of a VP16 TD, a GAL4 TD, an NF-κB TD,a BP64 TD, and a B42 acidic TD.

In one embodiment, a DNA binding domain, the ligand binding domain thatbinds an activating ligand, a ligand binding domain that dimerizes withthe ligand binding domain that binds an activating ligand, and atransactivation domain are encoded by polynucleotide sequences that arecontained in the same polynucleotide.

In another embodiment, a DNA binding domain, a ligand binding domainthat binds an activating ligand, a ligand binding domain that dimerizeswith the ligand binding domain that binds an activating ligand, and atransactivation domain are encoded by polynucleotide sequences that arecontained in two or more separate polynucleotide sequences.

In another embodiment, a DNA binding domain, a ligand binding domainthat binds an activating ligand, a ligand binding domain that dimerizeswith the ligand binding domain that binds an activating ligand, and atransactivation domain are encoded by polynucleotide sequences that arecontained in two separate polynucleotide sequences.

In another embodiment, a DNA binding domain and a ligand binding domainthat binds an activating ligand are encoded by polynucleotide sequencesthat are contained in a first polynucleotide sequence, and a ligandbinding domain that dimerizes with the ligand binding domain that bindsan activating ligand and a transactivation domain are encoded bypolynucleotide sequences that are contained in a second polynucleotidesequence.

In another embodiment, a DNA binding domain and a ligand binding domainthat dimerizes with the ligand binding domain that binds an activatingligand are encoded by polynucleotide sequences that are contained in afirst polynucleotide sequence, and a ligand binding domain that binds anactivating ligand and a transactivation domain are encoded bypolynucleotide sequences that are contained in a second polynucleotidesequence.

In embodiments in which one or more of the DNA binding domain, a ligandbinding domain that binds an activating ligand, a ligand binding domainthat dimerizes with the ligand binding domain that binds an activatingligand, and a transactivation domain are encoded by polynucleotidesequences that are contained in one or more separate polynucleotidesequences, then the one or more separate polynucleotide sequences areoperably linked to one or more separate promoters. In anotherembodiment, the one or more separate polynucleotide sequences areoperably linked to one or more separate enhancer elements. In anotherembodiment, the promoter(s) and/or the enhancer(s) are constitutivelyactive. In another embodiment, the promoter(s) and/or the enhancer(s)are tissue specific promoters and/or enhancers.

In one embodiment, the gene switch comprises a DNA binding domain, anecdysone receptor ligand binding domain, a ligand binding domain thatdimerizes with the ecdysone receptor ligand binding domain, and atransactivation domain.

In another embodiment, the gene switch comprises a DNA binding domain,an ecdysone receptor ligand binding domain, a retinoid X receptor ligandbinding domain, and a transactivation domain.

In another embodiment, the gene switch comprises a DNA binding domain,an ecdysone receptor ligand binding domain, a chimericvertebrate/invertebrate retinoid X receptor ligand binding domain, and atransactivation domain.

In another embodiment, the gene switch comprises a GAL4 DNA bindingdomain, a Choristoneura fumiferana ecdysone receptor ligand bindingdomain that is engineered to contain the mutations V107I and Y127E ofthe Choristoneura fumiferana ecdysone receptor sequence set forth in SEQID NO:1, a chimeric Homo sapiens/Locusta migratoria retinoid X receptorligand binding, and a VP16 transactivation domain.

In another embodiment, the host cell further comprises a polynucleotideencoding a peptide, protein or polypeptide whose expression is regulatedby the gene switch. A promoter that binds the gene switch complex isoperably linked to the polynucleotide encoding a peptide, protein orpolypeptide whose expression is regulated by the gene switch.

In another embodiment, the polynucleotide encoding a peptide, protein orpolypeptide whose expression is regulated by the gene switch iscontained in the same polynucleotide as a polynucleotide that encodesone or more of a DNA binding domain, the ligand binding domain thatbinds an activating ligand, a ligand binding domain that dimerizes withthe ligand binding domain that binds an activating ligand, and atransactivation domain. Such constructs are disclosed, for example, inU.S. Patent Publication No. 2009/0123441 (see also, WO 2009-048560(PCT/USUS2008/011563)).

In another embodiment, the polynucleotide encoding a peptide, protein orpolypeptide whose expression is regulated by the gene switch iscontained in a different nucleic acid molecule than a nucleic acidmolecule that encodes one or more of a DNA binding domain, the ligandbinding domain that binds an activating ligand, a ligand binding domainthat dimerizes with the ligand binding domain that binds an activatingligand, and a transactivation domain.

In one embodiment, the gene switch is more sensitive to an activatingligand than to a steroid hormone. In another embodiment, the gene switchis more sensitive to an activating ligand than to anotherdiacylhydrazine compound.

The sensitivity of a gene switch to an activating ligand, relative toanother ligand, can readily be determined in an in vitro assay, forexample, an in vitro assay that employs a reporter gene, such as fireflyluciferase. Examples of such in vitro assays are well known to those ofordinary skill in the art. See, for example, Karzenowski et al.,BioTechniques 39: 191-200 (2005).

In one embodiment, the polynucleotide encoding the gene switch iscontained in a vector. In one embodiment, the vector selected from thegroup consisting of a plasmid, an expression vector, a replicon, a phagevector, a cosmid, a viral vector, a liposome, an electrically chargedlipid (e.g., a cytofectin), a DNA-protein complex, and a biopolymer.

In another embodiment, the vector is a retroviral vector. In anotherembodiment, the vector is selected from the group consisting of anadeno-associated viral vector, a pox viral vector, a baculoviral vector,a vaccinia viral vector, a herpes simplex viral vector, an Epstein-Barrviral vector, an adenoviral vector, a gemini viral vector, and a caulimoviral vector.

In one embodiment, a composition of the invention comprises one or morepolynucleotides that encode two or more orthogonal gene switches. Two ormore individually operable gene regulation systems are said to be“orthogonal” when (a) modulation of each of the given gene switches byits respective ligand results in a measurable change in the magnitude ofexpression of the gene that is regulated by that gene switch, and (b)the change is statistically significantly different than the change inexpression of all other gene switches that are in the host cell. In oneembodiment, regulation of each individually operable gene switch systemeffects a change in gene expression at least 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 50-fold, 70-fold, 100-fold, 200-fold, 300fold, 400-fold or 500-fold greater than all of the other operable geneswitches in the host cell. Non-limiting examples of orthogonal geneswitch systems are set forth in U.S. Pat. No. 8,105,825 (Publication No.US 2002/0110861 A1).

As used herein, an “activating ligand” is a compound that bindsselectively to the ligand binding domain of a gene switch.

In one embodiment, the activating ligand is administered to the subjectwithin an hour of the time at which the priming dosage is administeredto the subject. In another embodiment, the activating ligand isadministered to the subject within about 24, 48, 96, 120, 144 or 168hours of the time at which the priming dosage is administered to thesubject. In another embodiment, the activating ligand is administered tothe subject within about 1, 2, 3, 4 or 5 weeks of the time at which thepriming dosage is administered to the subject.

In one embodiment, the activating ligand is administered to the subjectwithin an hour of the time at which the first of the at least oneboosting dosage is administered to the subject. In another embodiment,the activating ligand is administered to the subject within about 24,48, 96, 120, 144 or 168 hours of the time at which the first of the atleast one boosting dosage is administered to the subject. In anotherembodiment, the activating ligand is administered to the subject withinabout 1, 2, 3, 4 or 5 weeks of the time at which the first of the atleast one boosting dosage is administered to the subject.

In another embodiment, a composition of the invention is containedwithin a container. In one embodiment, the container is a vial. Inanother embodiment the container is a multiple-use vial. In anotherembodiment, the container displays an expiration date for thecomposition. In another embodiment, the container contains instructionsfor using the composition.

In one embodiment, a composition of the invention is a unit dosagecomposition. In one embodiment, a unit dosage composition is acomposition that is manufactured to supply a single dosage of thecomposition of the invention. In another embodiment, the unit dosagecomposition is manufactured to provide more than one measured dosages ofthe composition of the invention.

The present application also provides an article of manufacturecomprising more than one of the unit dosage compositions of theinvention. In one embodiment, the article of manufacture is a container.In another embodiment, the article of manufacture is a box. In anotherembodiment, the article of manufacture displays an expiration date forthe unit dosage composition.

The present invention also provides a kit comprising more than one ofthe composition or unit dosage of the present invention. In oneembodiment, the kit displays an expiration date for the composition orunit dosage. In another embodiment, the kit displays and/or or containsinstructions for using the composition or unit dosage. In anotherembodiment, the kit also comprises an activating ligand that binds tothe ligand binding domain of the gene switch encoded by thepolynucleotide in the composition or unit dosage.

The present invention also provides a drug label for the composition orunit dosage of the present invention. In one embodiment, the drug labeldisplays an expiration date for the composition or unit dosage. Inanother embodiment, the drug label displays instructions for using thecomposition or unit dosage. In another embodiment, the drug labeldisplays the approved indication(s) for the composition or unit dosage.In another embodiment, the said label is in paper form. In anotherembodiment, the drug label is in digital or computer-readable form.

The term “activating ligand” as used herein refers to a compound thatshows activity as an ecdysone receptor agonist, i.e., a compound that isable to mimic 20-hydroxyecdysone biological activity, and binds to agene switch ligand binding domain. Activating ligands for use in thepresent invention include both ecdysteroids and non-steroidal compounds,e.g., tebufenozide and methoxyfenozide.

In one embodiment, the activating ligand is an ecdysone receptor agonistdisclosed in U.S. Pat. No. 8,076,517 (Publication No. 2009/0163592), No.2009/0298175, No. 2005/0228016 and in U.S. Pat. No. 6,258,603, U.S. Pat.No. 7,375,093, No. 7,456,315, No. 7,304,161, and No. 7,304,162; each ofwhich are hereby incorporated by reference herein.

In certain embodiments, the activating ligand is a compound havingFormula I:

wherein:

A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;

B is optionally substituted aryl or optionally substituted heteroaryl;

E is CR¹R²R³;

R¹ is optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted heterocycle,optionally substituted aryl or optionally substituted heteroaryl; and

R² and R³ are independently hydrogen, optionally substituted alkyl,arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted heterocycle, optionally substituted aryl oroptionally substituted heteroaryl; or

R¹ and R² taken together form an optionally substituted alkenyl group.

In one embodiment the activating ligand is a compound having Formula I:

wherein:

A is selected from the group consisting of 2,3,6-tri-F-phenyl-;2,3-di-CH₃-phenyl-; 2,6-di-F-phenyl-; 2-Br, 3,4-ethylenedioxy-phenyl-;2-CH═CH₂, 3-OCH₃-phenyl-; 2-CH₂CH₃, 3,4-ethylenedioxy-phenyl-; 2-CH₂CH₃,3-OCH₃-phenyl-; 2-CH₂Cl, 3-OCH₃-phenyl-; 2-CH₂F, 3-OCH₃-phenyl-;2-CH₂NHCH₃, 3-OCH₃-phenyl-; 2-CH₂NMe₂, 3-OCH₃-phenyl-; 2-CH₂OAc,3-OCH₃-phenyl-; 2-CH₂OCH₂CH═CH₂, 3-OCH₃-phenyl-; 2-CH₂OH,3-OCH₃-phenyl-; 2-CH₂OMe, 3-OCH₃-phenyl-; 2-CH₂OMe, 3-OMe-phenyl-;2-CH₂S(O)₂CH₃, 3-OCH₃-phenyl-; 2-CH₂S(O)CH₃, 3-OCH₃-phenyl-; 2-CH₂SCH₃,3-OCH₃-phenyl-; 2-CH₃, 3,4-ethylenedioxy-phenyl-; 2-CH₃,3,4-OCH₂O-phenyl-; 2-CH₃, 3-Ac-phenyl-; 2-CH₃, 3-CH₂CH₂CH₂O-4-phenyl-;2-CH₃, 3-CH₃-phenyl-; 2-CH₃, 3-Cl-phenyl-; 2-CH₃, 3-Et-phenyl-; 2-CH₃,3-I-phenyl-; 2-CH₃, 3-NMe₂-phenyl-; 2-CH₃, 3-NO₂-phenyl-; 2-CH₃,3-OAc-phenyl-; 2-CH₃, 3-OCF₃-phenyl-; 2-CH₃, 3-OCH₂OCH₂-4-phenyl-;2-CH₃, 3-OCH₃-phenyl-; 2-CH₃, 3-OH-phenyl-; 2-CH₃, 3-Oi-Pr-phenyl-;2-CH₃, 3-OMe-phenyl-; 2-CH₃, 4,5-methylenedioxy-phenyl-;2-CH₃-3-OCH₃-phenyl-; 2-Cl 4,5-methylenedioxy-phenyl-; 2-Cl,3-CH₂OCH₂O-4-phenyl-; 2-Cl, 3-CH₂OCH₂O-4-phenyl-; 2-Cl, 3-OMe-phenyl-;2-Et, 3,4-ethylenedioxy-phenyl-; 2-Et, 3,4-OCH(CH₃)O-phenyl-; 2-Et,3,4-OCH₂O-phenyl-; 2-Et, 3-OCH₃-phenyl-2-F, 3,4-CH₂OCH₂O-phenyl-; 2-F,4-CH₂CH₃-phenyl-; 2-F, 4-Et-phenyl-; 2-I, 3-OMe-phenyl-; 2-NH₂,3-OMe-phenyl-; 2-NO₂, 3-OMe-phenyl-; 2-Vinyl, 3-OMe-phenyl-;3,4-(CH₂)₄-phenyl-; 3,4-di-Et-phenyl-; 3,4-ethylenedioxy-phenyl-;3,4-OCF₂O-phenyl-; 3,4-OCH(CH₃)O-phenyl-; 3,4-OCH₂O-phenyl-; 3-Cl,4-Et-phenyl-; 3-NH—C═C-4-phenyl-; 3-OCH(CH₃)CH₂O-4-phenyl-; 3-OCH₃,4-CH₃-phenyl-; 3,4-S—C═N-phenyl-; 4-Br-phenyl-; 4-C(O)CH₃-phenyl-;4-CH(OH)CH₃-phenyl-; 4-CH₂CH₃-phenyl-; 4-CH₂CN-phenyl-; 4-CH₃-phenyl-;4-Cl-phenyl-; 4-Et-phenyl-; 4-OCH₃-phenyl-phenyl-; andbenzo[1,2,5]oxadiazole-5-yl;

B is selected from the group consisting of 1-trityl-5-benzimidazolyl-;3-trityl-5-benzimidazolyl-; 1H-indazole-3-yl-; 1-methyl-1H-indole-2-yl-;1-methyl-2-oxo-6-trifluoromethyl-3-pyridyl-; 1-trityl-1H-indazole-3-yl-;2,3,4,5-phenyl-; 2,3,4,5-tetra-F-phenyl-; 2,3,4-F-phenyl-;2,3-F-phenyl-; 2,3-OCH₂O-phenyl-; 2,4,5-F-phenyl-;2,4-di-Cl-5-F-phenyl-; 2,5-di-OCH₃-phenyl-; 2,5-F-phenyl-;2,6-di-Cl-4-pyridyl-;2,6-dimethoxy-4-pyrimidinyl2,6-di-OCH₃-3-pyridyl2,6-F-phenyl-; 2-Cl,5-NO₂-phenyl-; 2-Cl-3-pyridyl2-Cl-4-F-phenyl-; 2-Cl-5-CH₃-phenyl-;2-Cl-6-CH₃-4-pyridyl-; 2-Et-phenyl-; 2-F, 4-Cl-phenyl-; 2-F,5-CH₃-phenyl-; 2-methoxy-6-trifluoromethyl-3-pyridyl-;2-NO₂-3,5-di-OCH₃, 4-CH₃-phenyl-; 2-NO₂-4-Cl-phenyl-;2-NO₂-5-CH₃-phenyl-; 2-NO₂-5-Cl-phenyl-; 2-NO₂-5-F-phenyl-;2-NO₂-phenyl-; 2-OCH₂CF₃, 5-OCH₃-phenyl-;2-OCH₃-3-pyridyl2-OCH₃-4-CH₃-phenyl-; 2-OCH₃-4-Cl-phenyl-;2-OCH₃-4-F-phenyl-; 2-OCH₃-5-CH₃-phenyl-; 2-OCH₃-5-Cl-phenyl-;2-OCH₃-phenyl-; 2-S(O)CH₃-phenyl-; 2-SO₃H-phenyl-; 3,4,5-F-phenyl-;3,4,5-tri-OCH₃-phenyl-; 3,4-di-CH₃-5-Cl-phenyl-; 3,4-F-phenyl-;3,4-methylenedioxy-phenyl-; 3,5-di(CH₂OH)-phenyl-;3,5-di-CH₃-4-Cl-phenyl-; 3,5-di-CH₃-phenyl-; 3,5-di-CI-4-F-phenyl-;3,5-di-Cl-phenyl-; 3,5-di-CO₂H-phenyl-; 3,5-di-F-phenyl-; 3,5-di-OCH₃,4-CH₃-phenyl-; 3,5-di-OCH₃-4-OAc-phenyl-; 3,5-di-OCH₃-phenyl-;3,6-dichloro-4-pyridazinyl-; 3,6-dimethoxy-4-pyridazinyl-; 3-Br-phenyl-;3-CF₃, 5-F-phenyl-; 3-CF₃-4-F-phenyl-; 3-CF₃-4-F-phenyl3-CF₃-phenyl-;3-CH═NNHCOCONH₂, 5-CH₃-phenyl-; 3-CH═NNHCONH₂, 5-CH₃-phenyl-; 3-CH═NOH,5-CH₃-phenyl-; 3-CH₂OAc, 5-CH₃-phenyl-; 3-CH₃, 5-Br-phenyl-; 3-CH₃,5-CH₃-phenyl-; 3-CH₃, 5-Cl-phenyl-; 3-CH₃-4-Br-phenyl-; 3-CH₃-phenyl-;3-chloro-6-methylsulfanyl-pyrazine-2-yl-; 3-Cl, 5-Br-phenyl-; 3-Cl,5-Cl-phenyl-; 3-Cl-5-OCH₃-4-pyridyl-; 3-Cl-phenyl-; 3-CN-phenyl-, 3-F,5-F-phenyl-; 3-F-phenyl-; 3-NO₂-phenyl-; 3-OCH₃-4-CH₃-phenyl-;3-OCH₃-4-pyridyl-; 3-OCH₃-phenyl-; 3-OMe, 5-CH₃-phenyl-; 3-OMe,5-OMe-phenyl-; 3-oxo-6-methoxy-4-pyridazinyl-; 4,6-dimethyl-pyridyl-;4-CH₃-phenyl-; 4-F-phenyl-; 4-pyridazinyl-; 5-benzimidazolyl-;5-methoxycarbonyl-2-pyridyl-; 5-methyl-1-phenyl-1H-pyrazole-3-yl-;5-methyl-pyrazine-2-yl-; 6-CH₃-2-pyridyl-; phenyl-; and pyrazine-2-yl;and

E is selected from the group consisting of C(CH₃)₂C(O)OEt;C(CH₃)₂CH═NCH₂CH₂OH; C(CH₃)₂CH═NNHC(O)C(O)NH₂; C(CH₃)₂CH═NNHC(O)NH₂;C(CH₃)₂CH═NOH; C(CH₃)₂CH₂OC(O)CH₃; C(CH₃)₂CH₂OCH₃; C(CH₃)₂CH₂OH;C(CH₃)₂CH₂OSi(CH₃)₂tBu; C(CH₃)₂CHO: C(CH₃)₂CN; C(CH₃)₂COOH;CH(CH₃)C(CH₃)₃; CH(Et)(n-Bu); CH(Et)(t-Bu;) CH(n-Bu)(t-Bu);CH(n-Pr)(t-Bu); CH(Ph)(t-Bu); and t-Bu.

In another embodiment, the activating ligand is a compound havingFormula I wherein A, B, and E are defined according to Table 3.

TABLE 3 Ligand Components A B E 4-Cl—Ph Ph t-Bu 4-Et—Ph 2-NO₂—Ph t-Bu4-CH₃—Ph 3-CH₃, 5-CH₃—Ph t-Bu 4-Et—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2,6-di-F—Ph3-Cl, 5-Cl—Ph t-Bu 2-CH₃, 3-Cl—Ph 3-Cl—Ph t-Bu 2-Cl, 3-OMe—Ph2-Cl-5-CH₃—Ph t-Bu 2-CH₃, 3-Cl—Ph 3-CH₃-4-Br—Ph t-Bu 4-Et—Ph3,5-di-CH₃-4-Cl—Ph t-Bu 4-Et—Ph 3,4-di-CH₃-5-Cl—Ph t-Bu 4-OCH₃—Ph2-Cl-4-F—Ph t-Bu 4-Et—Ph 3-CH₃, 5-Cl—Ph t-Bu 4-Et—Ph 2-Et—Ph t-Bu4-OCH₃—Ph 3-Cl, 5-Cl—Ph t-Bu 4-Et—Ph 2-NO₂-5-CH₃—Ph t-Bu 4-CH₂CN—Ph3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 3-CH₃—Ph t-Bu 4-Br—Ph 3-Cl, 5-Cl—Pht-Bu 2-CH₃, 3-NO₂—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-CH₃—Ph 2,5-di-OCH₃—Pht-Bu 2-CH₃, 3-CH₃—Ph 2-OCH₃-5-Cl—Ph t-Bu 2-NO₂, 3-OMe—Ph 3-CH₃, 5-CH₃—Pht-Bu 2-CH₃, 3-CH₃—Ph 3-OMe, 5-OMe—Ph t-Bu 3-Cl, 4-Et—Ph 3-CH₃, 5-CH₃—Pht-Bu 4-CH(OH)CH₃—Ph 3-F, 5-F—Ph t-Bu 2-CH₃, 3-NMe₂—Ph 3-Cl, 5-Cl—Ph t-Bu2-CH₃, 3-Ac—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OAc—Ph 3-CH₃, 5-CH₃—Ph t-Bu2-CH₃, 3-I—Ph 3-CH3, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 3-Cl, 5-Br—Ph t-Bu2-CH₃, 3-Oi-Pr—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OCH3—Ph 2-Cl-3-pyridylt-Bu 2-CH₃, 3-OMe—Ph 2-OCH₃-5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 2,5-F—Ph t-Bu2-CH₃, 3-OMe—Ph 2-Et—Ph t-Bu 2-CH₃, 3-OMe—Ph 3-CH₃, 5-Br—Ph t-Bu 2-CH₃,3-OMe—Ph 3-OMe, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 2-OCH₃-4-Cl—Ph t-Bu 2-CH₃,3-OCF₃—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 3-OCH₃-4-CH₃—Ph t-Bu3-OCH₃, 4-CH3—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 2-OCH₃-4-CH₃—Pht-Bu 2-CH₃, 3-OCH₃—Ph 2,6-di-Cl-4-pyridyl t-Bu 2-CH₃, 3-OMe—Ph2-NO₂-5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 2-F-4-Cl—Ph t-Bu 3,4-OCH₂O—Ph2-Cl-4-F—Ph t-Bu 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-Et—Ph3-CH₃, 5-CH₃—Ph t-Bu 3-CH₂CH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph3,5-di-Cl-4-F—Ph t-Bu 2-CH₃, 3,4-OCH₂O—Ph 4-F—Ph t-Bu 2-Et, 3,4-OCH₂O—Ph2-OCH₃—Ph t-Bu 3,4-di-Et—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 4-F—Pht-Bu 2-Et, 3-OMe—Ph 2-OCH₃—Ph t-Bu 2-CH₃, 3-OMe—Ph 2-OCH₃-4-F—Ph t-Bu2-Et, 3-OCH₃—Ph 2-Cl-6-CH₃-4-pyridyil t-Bu 2-Et, 3-OMe—Ph 3-OMe,5-OMe—Ph t-Bu 2-I, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph t-Bu 3,4-ethylenedioxy-Ph2-OCH₃—Ph t-Bu 3,4-(CH₂)₄—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph2,3-OCH₂O—Ph t-Bu 2-F, 4-Et—Ph 4-F—Ph t-Bu 2-Et, 3-OMe—Ph3,4-methylenedioxy-Ph t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph 4-F—Ph t-Bu3,4-OCH(CH₃)O—Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-Et, 3,4-OCH(CH₃)O—Ph 3-CH₃,5-CH₃—Ph t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph 3-OCH₃—Ph t-Bu3-OCH(CH₃)CH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-Br, 3,4-ethylenedioxy-Ph3-CH₃, 5-CH₃—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph 3-CH₃, 5-Cl—Ph t-Bu2-Et, 3,4-ethylenedioxy-Ph 3-CH₃—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph2-OCH₃—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph 3-OCH₃—Ph t-Bu 3-S—C═N-4-Ph3-CH₃, 5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 2-OCH₃-4-Cl—Ph t-Bu 2-Et, 3-OMe—Ph2,5-di-OCH₃—Ph t-Bu 2-CH₃, 4,5-methylenedioxy-Ph 3-CH₃, 5-CH₃—Ph t-Bu3-CH₂OCH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-CH₃, 3-OCH₂OCH₂-4-Ph 2-OCH₃—Pht-Bu 2-Et, 3-OCH₂OCH₂-4-Ph 4-F—Ph t-Bu 2-Cl 4,5-methylenedioxy-Ph 3-CH₃,5-CH₃—Ph t-Bu 2,3,6-tri-F—Ph 2-Cl-4-F—Ph t-Bu 2-Et, 3-OMe—Ph 2,6-F—Pht-Bu 2-Et, 3-OMe—Ph 3-F—Ph t-Bu 2-Et, 3-OMe—Ph 3-Br—Ph t-Bu 2-Et,3-OMe—Ph 2-NO₂—Ph t-Bu 2-Et, 3-OMe—Ph 2,3-F—Ph t-Bu 2-Et, 3-OMe—Ph3,4,5-tri-OCH₃—Ph t-Bu 2-Et, 3-OMe—Ph 3-CF₃, 5-F—Ph t-Bu 2-Et, 3-OMe—Ph3-CN—Ph t-Bu 2-Vinyl, 3-OMe—Ph 2,4-di-Cl-5-F—Ph t-Bu 2-Et,3-OCH₂OCH₂-4-Ph Ph t-Bu 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂C(O)OEt2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CH₂OH 2-Et, 3-OMe—Ph 3-CH₃,5-CH₃—Ph —C(CH₃)₂CHO 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CH₂OCH₃2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CH═NOH 2-NH₂, 3-OMe—Ph 3-CH₃,5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 3-CH₂OAc, 5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph3-CH₃, 5-CH₃—Ph —C(CH₃)₂CH₂OC(O)CH₃ 2-CH₃, 3-OH—Ph 2,3,4-F—Ph t-Bu2-CH₃, 3-OH—Ph 3-Cl-5-OCH₃-4-pyridyl t-Bu 2-CH₃, 3-OH—Ph2,6-di-Cl-4-pyridyl t-Bu 2-CH₃, 3-OH—Ph 3-OCH₃-4-pyridyl t-Bu 2-CH₃,3-OH—Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 2-OCH₃—Pht-Bu 2-Et, 3-OMe—Ph 2,4-di-Cl-5-F—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph2,4-di-Cl-5-F—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 2-F, 5-CH₃—Ph t-Bu 2-CH₃,3-CH₂CH₂CH₂O-4-Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph2,5-Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph 2,3,4-F—Ph t-Bu 2-Et,3,4-ethylenedioxy-Ph 2,3,4,5-Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph3-CF₃-4-F—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph 2,6-di-Cl-4-pyridyl t-Bu2-Et, 3,4-ethylenedioxy-Ph 2-OCH₃—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph2,4-di-Cl-5-F—Ph t-Bu 2-Et, 3,4-ethylenedioxy-Ph 2-F, 4-Cl—Ph t-Bu2-CH₃, 3-OAc—Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 2-OCH₃-5-Cl—Pht-Bu 2-Et, 3,4-OCH₂O—Ph 2-OCH₃-4-Cl—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph2-OCH₃-5-Cl—Ph t-Bu 2-Et, 3-OMe—Ph 2-NO₂-5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph2-NO₂-4-Cl—Ph t-Bu 2-Et, 3-OMe—Ph 2-NO₂-5-Cl—Ph t-Bu 2-CH₃,3-CH₂CH₂CH₂O-4-Ph 2-NO₂-5-CH₃—Ph t-Bu Benzo[1,2,5]oxadiazole-5-yl2-OCH₃-4-Cl—Ph t-Bu 2-Vinyl, 3-OMe—Ph 2-Cl, 5-NO₂—Ph t-Bu 2-Vinyl,3-OMe—Ph 2-OCH₃-4-Cl—Ph t-Bu 2-Et, 3-OCH₃—Ph 1-methyl-1H-indole-2-ylt-Bu 2-Et, 3,4-ethylenedioxy-Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu 2-Cl,3-CH₂OCH₂O-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu 2-F, 4-Et—Ph 3-NO₂—Ph t-Bu 2-F,4-Et—Ph 3-OCH₃—Ph t-Bu 2-Cl, 3-CH₂OCH₂O-4-Ph 3,5-di-OCH₃-4-CH₃—Ph t-Bu2-F, 4-Et—Ph 2,6-di-Cl-4-pyridyl t-Bu 2-F, 4-Et—Ph 3,5-di-OCH₃-4-CH₃—Pht-Bu 2-F, 4-Et—Ph 3,4,5-F—Ph t-Bu 2-F, 4-Et—Ph 3-CH₃—Ph t-Bu 2-F,4-Et—Ph 2-OCH₃—Ph t-Bu 2-F, 4-Et—Ph 2-NO₂-5-F—Ph t-Bu 2-F, 4-Et—Ph2-OCH₂CF₃, 5-OCH₃—Ph t-Bu 2-F, 4-Et—Ph 2-Cl-6-CH₃-4-pyridyl t-Bu 2-F,4-Et—Ph 2,6-di-OCH₃-3-pyridyl t-Bu 3-NH—C═C-4-Ph 3-CH₃, 5-CH₃—Ph t-Bu2-Et, 3-OMe—Ph 2-S(O)CH₃—Ph t-Bu 3,4-OCF₂O—Ph 2-NO₂—Ph t-Bu 3,4-OCF₂O—Ph3-CH₃, 5-CH₃—Ph t-Bu 3,4-OCF₂O—Ph 3-OCH₃—Ph t-Bu 2-Et, 3-OMe—Ph 3-Br—Ph—C(CH₃)₂CN 2-CH₂OMe, 3-OMe—Ph 3,5-di-Cl—Ph t-Bu 2-Et, 3-OMe—Ph 3-CH═NOH,5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 3-CH═NNHCONH₂, 5-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph3-CH═NNHCOCONH₂, t-Bu 5-CH₃—Ph 2-Et, 3-OMe—Ph 3-CH₃, 5-CH₃—Ph —C(CH₃)₂CN2-Et, 3-OMe—Ph 3,5-di-OCH₃-4-CH₃—Ph —C(CH₃)₂CN 2-Et, 3-OCH₂OCH₂-4-Ph2-OCH₃—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 2,4,5-F—Ph t-Bu 2-CH₃,3-CH₂CH₂CH₂O-4-Ph 3,4,5-F—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 3-F—Ph t-Bu2-Et, 3,4-OCH₂O—Ph 3-CF₃—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 4-F—Ph t-Bu2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 3,4-F—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph3,5-di-F—Ph t-Bu 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph 2,3,4,5-tetra-F—Ph t-Bu 2-Et,3-OCH₂OCH₂-4-Ph 4-CH₃—Ph t-Bu 2-Et, 3-OMe—Ph 3,5-di-OCH₃-4-OAc—Ph t-Bu2-Et, 3-OMe—Ph 3,5-di-OCH₃—OH—Ph t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph3,5-di-OCH₃-4-CH₃—Ph t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph2,6-di-OCH₃-3-pyridyl t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph2,6-di-Cl-4-pyridyl t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph 3-F—Ph t-Bu 2-CH₃,3,4-ethylenedioxy-Ph 3-CF₃, 5-F—Ph t-Bu 2-CH₃, 3,4-ethylenedioxy-Ph2-NO₂-5-CH₃—Ph t-Bu 2-ethyl, 3-methoxy 4,6-dimethyl-pyridyl t-Bu 2-CH₃,3,4-ethylenedioxy-Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₃,3,4-ethylenedioxy-Ph 3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₃,3,4-ethylenedioxy-Ph 3,5-di-CH₃—Ph —CH(n-Pr)C(CH₃)₃ 2-CH₃,3,4-ethylenedioxy-Ph 3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃ 2-CH₂CH₃,3,4-ethylenedioxy-Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₂CH₃,3,4-ethylenedioxy-Ph 3,5-di-OCH3-4-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₂CH₃,3,4-ethylenedioxy-Ph 3,5-di-CH₃-3-Ph —CH(n-Pr)C(CH₃)₃ 2-CH₂CH₃,3,4-ethylenedioxy-Ph 3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃ 2-CH₃,3-OCH₃—Ph 2-methoxy-6-trifluoromethyl-3- —C(CH₃)₃ pyridyl 2-CH3,3-OCH₃—Ph 1-methyl-2-oxo-6- —C(CH₃)₃ trifluoromethyl-3-pyridyl 2-CH₃,3-OCH₃—Ph 2,6-dimethoxy-4-pyrimidinyl —C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph3,6-dimethoxy-4-pyridazinyl —C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph3,6-dichloro-4-pyridazinyl —C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph 4-pyridazinyl—C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph 3-oxo-6-methoxy-4-pyridazinyl —C(CH₃)₃ 2-CH₃,3-OCH₃—Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph—CH(n-Pr)C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph 3,5-di-OCH₃-4-CH₃—Ph —CH(n-Pr)C(CH₃)₃2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-OCH₃-4-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph—CH(n-Pr)C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-OCH₃-4-CH₃—Ph—CH(n-Pr)C(CH₃)₃ 2-CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —CH(Et)C(CH₃)₃ 2-CH₃,3-OH—Ph 3-OCH₃-4-pyridyl —C(CH₃)₃ 4-CH(OH)CH₃—Ph 3,5-di(CH₂OH)—Ph—C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 2-S(O)CH₃—Ph —C(CH₃)₃ 4-C(O)CH₃—Ph3,5-di-CO₂H—Ph —C(CH₃)₃ 2-CH₃, 3,4-ethylenedioxy-Ph2,6-di-OCH₃-3-pyridyl —C(CH₃)₃ 2-CH₂CH₃, 3,4-ethylenedioxy-Ph3-CF₃-4-F-phenyl —C(CH₃)₃ 2-F, 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃2-CH₂CH₃, 3-OCH₃—Ph 2-SO₃H—Ph —C(CH₃)₃ 2-CH₃, 3-CH₂CH₂CH₂O-4-Ph3,5-di-CH₃—Ph —C(CH₃)₃ 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —CH(CH₃)C(CH₃)₃2-CH₂CH₃, 3,4-ethylenedioxy-Ph 3-CH₃—Ph —C(CH₃)₃ 2,3-di-CH₃—Ph Ph—CH(Et)(n-Bu) 2,3-di-CH₃—Ph 3-CH₃—Ph —CH(Et)(t-Bu) 2-CH₃,3,4-ethylenedioxy-Ph 3,5-di-OCH₃, 4-OH—Ph —C(CH₃)₃ 2-F, 3-CH₂OCH₂O-4-Ph3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₃, 3,4-ethylenedioxy-Ph 2-S(O)CH₃—Ph —C(CH₃)₃2-CH₃, 3,4-ethylenedioxy-Ph 3,5-di-OCH₃, 4-CH₃—Ph —C(CH₃)₂CN2-CH₂CH₃-3-OCH₃—Ph 6-CH₃-2-pyridyl- —C(CH₃)₃ 2-CH₃, 3,4-ethylenedioxy-Ph2-NO₂-di-OCH₃, 4-CH₃—Ph —C(CH₃)₃ 2-CH₃, 3,4-ethylenedioxy-Ph3,5-di-CH₃—Ph —C(CH₃)₃ 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃2-CH₃-3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ 4-CH₂CH₃—Ph 3,5-di-CH₃—Ph—CH(Et)(t-Bu) 4-CH₂CH₃—Ph 2-OCH₃-3-pyridyl —CH(Et)(t-Bu) 4-CH₂CH₃—Ph3,5-di-CH₃—Ph —CH(n-Bu)(t-Bu) 4-CH₂CH₃—Ph 3,5-di-OCH₃, 4-CH₃—Ph—CH(n-Bu)(t-Bu) 4-CH₂CH₃—Ph 2-OCH₃-3-pyridyl —CH(n-Bu)(t-Bu) 4-CH₂CH₃—Ph3,5-di-CH₃—Ph —CH(Ph)(t-Bu) 4-CH₂CH₃—Ph 3,5-di-OCH₃, 4-CH₃—Ph—CH(Ph)(t-Bu) 4-CH₂CH₃—Ph 2-OCH₃-3-pyridyl —CH(Ph)(t-Bu) 2-CH₂CH₃,3-OCH₃—Ph 5-benzimidazolyl —C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 1-(or3-)trityl-5-benzimidazolyl —C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph5-methyl-1-phenyl-1H-pyrazole- —C(CH₃)₃ 3-yl 2-CH₂CH₃, 3-OCH₃—Ph3-chloro-6-methylsulfanyl- —C(CH₃)₃ pyrazine-2-yl 2-CH₂CH₃, 3-OCH₃—Ph1H-indazole-3-yl —C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 1-trityl-1H-indazole-3-yl—C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph 5-methoxycarbonyl-2-pyridyl —C(CH₃)₃2-CH₂CH₃, 3-OCH₃—Ph pyrazine-2-yl —C(CH₃)₃ 2-CH₂CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —C(CH₃)₂CH₂OSi(CH₃)2tBu 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph—C(CH₃)₂CH═NCH₂CH₂OH 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph—(CH₃)₂CH═NNHC(O)NH₂ 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph—C(CH₃)₂CH═NNHC(O)C(O)NH₂ 2-CH₂CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₂COOH2-CH₂S(O)CH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂S(O)₂CH₃, 3-OCH₃—Ph3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂NMe₂, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃2-CH₂NHCH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH═CH₂, 3-OCH₃—Ph—3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂OMe, 3-OCH₃—Ph— 3,5-di-CH₃—Ph —C(CH₃)₃2-CH₂SCH₃, 3-OCH₃—Ph 3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂OCH₂CH═CH₂,3,5-di-CH₃—Ph —C(CH₃)₃ 3-OCH₃—Ph 2-CH₂Cl, 3-OCH₃—Ph— 3,5-di-CH₃—Ph—C(CH₃)₃ 2-CH₂OH, 3-OCH₃—Ph— 3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂OAc, 3-OCH₃—Ph3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₂F, 3-OCH₃—Ph— 3,5-di-CH₃—Ph —C(CH₃)₃ 2-CH₃,3-OCH₃ 3,5-di-CH₃—Ph —CH(n-Bu)(t-Bu) 2-CH₃, 3-OCH₃ 3,5-di-OCH₃, 4-CH₃—CH(n-Bu)(t-Bu) 2-CH₂CH₃, 3-OCH₃—Ph 5-Methyl-pyrazine-2-yl- —C(CH₃)₃

In another embodiment, the activating ligand is a compound havingFormula I selected from the group consisting of:

-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-hydroxymethyl-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N-[3-(tert-butyl-dimethyl-silanyloxymethyl)-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl]1-hydrazide;-   7-[N′-tert-Butyl-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic    acid;-   7-[N′-tert-Butyl-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic    acid methyl ester,-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-semicarbazidomethyl-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   Phenyl-carbamic acid    7-[N′-tert-butyl-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl    ester;-   3,5-Dimethyl-benzoic acid    N-[3-(2-amino-ethyl)-5-methyl-2,3-dihydro-benzo[1.4]dioxine-6-carbonyl]-N-tert-butyl-hydrazide;-   7-[N′-tert-Buty    l-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1.4]dioxine-2-carboxylic    acid pentafluorophenyl ester;-   7-[N′-tert-Butyl-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic    acid methylamide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-formyl-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   Toluene-4-sulfonic acid    7-[N′-tert-butyl-N′-(3,5-dimethyl-benzoyl)-hydrazinocarbonyl]-8-methyl-2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl    ester;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-[3-hydroxyimino-methyl)-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl]-hydrazide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-cyanomethyl-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(5-methyl-3-methylsulfanylmethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-methanesulfonylmethyl-5-methyl-2,3-dihydro-benzo[1.4]dioxine-6-carbonyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-tert-butyl-N′-(3-fluoromethyl-5-methyl-2,3-dihydro-benzo[1.4]dioxine-6-carbonyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-heptyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide;-   3,5-Dimethoxy-4-methyl-benzoic    acid-N-(1-tert-butyl-heptyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   3,5-Dimethoxy-4-methyl-benzoic    acid-N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide;-   2-Methoxy-nicotinic acid    N-(1-tert-butyl-heptyl)-N′-(4-ethyl-benzoyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-3,4,4-trimethyl-pent-2-enyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-2-cyano-vinyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   3,5-Dimethyl-benzoic acid    N-(1-butyl-2,2-dimethyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;    and-   3,5-Dimethyl-benzoic acid    N-(1-butyl-2,2-dimethyl-pent-4-enyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

In another embodiment, the activating ligand is an enantiomericallyenriched compound having Formula II:

wherein:

-   -   A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally        substituted aryl or optionally substituted heteroaryl;    -   B is optionally substituted aryl or optionally substituted        heteroaryl; and    -   R¹ and R² are independently optionally substituted alkyl,        arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted        cycloalkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted heterocycle,        optionally substituted aryl or optionally substituted        heteroaryl;    -   with the proviso that R¹ does not equal R²;    -   wherein the absolute configuration at the asymmetric carbon atom        bearing R¹ and R² is predominantly S.

In another embodiment, the activating ligand is an enantiomericallyenriched compound having Formula III:

wherein:

-   -   A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally        substituted aryl or optionally substituted heteroaryl;    -   B is optionally substituted aryl or optionally substituted        heteroaryl; and    -   R¹ and R² are independently optionally substituted alkyl,        arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted        cycloalkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted heterocycle,        optionally substituted aryl or optionally substituted        heteroaryl;    -   with the proviso that R¹ does not equal R²;    -   wherein the absolute configuration at the asymmetric carbon atom        bearing R¹ and R² is predominantly R.

In another embodiment, the activating ligand is an enantiomericallyenriched compound having Formula III, wherein:

-   -   A is:

-   -   B is:

-   -   R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g), R^(3h),        R^(3i) and R^(3j) are independently selected from hydrogen,        halo, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy;    -   R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl, or (C₂-C₄)alkenyl; and    -   R² is optionally substituted (C₁-C₆)alkyl.

In another embodiment, the activating ligand is a compound havingFormula III selected from the group consisting of:

-   (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid benzyl ester;-   (R)—N′-(1-tert-Butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazinecarboxylic    acid tert-butyl ester;-   (R)—N′-(1-tert-Butyl-4-hydroxy-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazine    carboxylic acid benzyl ester;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-chloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,4-dimethoxy-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-difluoro-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzo[1,3]dioxole-5-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-1-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-2-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide;-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;-   (R)-3,5-Dimethoxy-4-methyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;    and-   (R)-3,5-Dimethyl-benzoic acid    N′-(4-ethyl-benzoyl)-N-(1-phenethyl-but-3-enyl)-hydrazide.

In another embodiment, the activating ligand is a compound havingFormula IV:

wherein:

-   -   Q is O or S;    -   R¹ is selected from the group consisting of hydrogen,        (C₁-C₁₂)alkyl, (C₃-C₁₂)cycloalkyl,        (C₃-C₁₂)cycloalkyl(C₁-C₃)alkyl, (C₁-C₁₂)haloalkyl,        (C₂-C₁₂)alkenyl, (C₃-C₁₂)cycloalkenyl, (C₂-C₁₂)haloalkenyl,        (C₂-C₁₂)alkynyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,        (C₁-C₆)alkylthio(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonyl,        succinimidylmethyl, benzosuccinimidylmethyl, optionally        substituted phenyl, optionally substituted 1-naphthyl,        optionally substituted 2-naphthyl, optionally substituted        phenyl(C₁-C₃)alkyl, optionally substituted phenyl(C₂-C₃)alkenyl,        optionally substituted naphthyl(C₁-C₃)alkyl, optionally        substituted phenoxy(C₁-C₃)alkyl, optionally substituted        phenylamino, and optionally substituted heterocycle;    -   R² and R³ are each independently selected from hydrogen,        (C₁-C₆)alkyl, and (C₁-C₆)haloalkyl;    -   R⁴ is hydrogen, (C₁-C₆)alkyl, or (C₁-C₆)haloalkyl;    -   R⁵ and R⁶ are each independently selected from the group        consisting of hydrogen, (C₁-C₁₂)alkyl, (C₃-C₁₂)cycloalkyl,        (C₁-C₁₂)haloalkyl, (C₂-C₁₂)alkenyl, (C₃-C₁₂)cycloalkenyl,        (C₂-C₁₂)haloalkenyl, (C₂-C₁₂)alkynyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,        (C₁-C₆)alkylthio(C₁-C₆)alkyl, aminocarbonyl, aminothiocarbonyl,        formyl, (C₁-C₆)alkylsulfinyl, (C₁-C₆)alkylsulfonyl,        (C₁-C₆)alkylcarbonyl, cyclo(C₃-C₆)alkylcarbonyl,        halo(C₁-C₆)alkylcarbonyl, (C₁-C₆)alkylaminocarbonyl,        di(C₁-C₆)alkylaminocarbonyl, (C₁-C₆)alkoxycarbonyl,        (C₁-C₆)alkoxycarbonylcarbonyl, or phenyl(C₂-C₃)alkenylcarbonyl,        optionally substituted phenyl, optionally substituted        1-naphthyl, optionally substituted 2-naphthyl, optionally        substituted phenyl(C₁-C₃)alkyl, optionally substituted        phenyl(C₂-C₃)alkenyl, optionally substituted phenylcarbonyl, or        optionally substituted heterocycle;    -   R⁷, R⁸, R⁹, and R¹⁰ are each independently selected from the        group consisting of hydrogen, cyano, nitro, halogen,        (C₁-C₁₂)alkyl, (C₃-C₁₂)cycloalkyl, (C₁-C₁₂)haloalkyl,        (C₂-C₁₂)alkenyl, (C₃-C₁₂)cycloalkenyl, (C₂-C₁₂)haloalkenyl,        (C₂-C₁₂)alkynyl, halo(C₂-C₆)alkynyl, hydroxy, (C₁-C₆)alkoxy,        halo(C₁-C₆)alkoxy, (C₂-C₆)alkenyloxy, halo(C₂-C₆)alkenyloxy,        (C₂-C₆)alkynyloxy, halo(C₂-C₆)alkynyloxy, aryloxy,        (C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)alkylthio,        halo(C₁-C₆)alkylthio, (C₂-C₆)alkenylthio,        halo(C₂-C₆)alkenylthio, (C₂-C₆)alkynylthio,        halo(C₂-C₆)alkynylthio, (C₁-C₆)alkylsulfinyl,        halo(C₁-C₆)alkylsulfinyl, (C₁-C₆)alkylsulfonyl,        halo(C₁-C₆)alkylsulfonyl, (C₁-C₆)alkylamino,        di(C₁-C₆)alkylamino, (C₁-C₃)alkoxy(C₁-C₃)alkyl,        (C₁-C₆)alkylthio(C₁-C₆)alkyl, (C₁-C₃)alkylsulfinyl(C₁-C₃)alkyl,        (C₁-C₃)alkylsulfonyl(C₁-C₃)alkyl, (C₁-C₃)alkylamino(C₁-C₃)alkyl,        di(C₁-C₃)alkylamino(C₁-C₃)alkyl, (C₁-C₆)alkylcarbonyl,        (C₁-C₆)alkylaminocarbonyl, di(C₁-C₆)alkylaminocarbonyl, or        (C₁-C₆)alkoxycarbonyl, optionally substituted phenyl, optionally        substituted 1-naphthyl, optionally substituted 2-naphthyl,        optionally substituted phenyl(C₁-C₃)alkyl, optionally        substituted phenyl(C₂-C₃)alkenyl, or optionally substituted        heterocycle.

In another embodiment, the activating ligand is a compound havingFormula IV wherein:

-   -   Q is O;    -   R¹ is selected from the group consisting of 4-fluorophenyl,        3-fluorophenyl, 4-fluoro-3-methylphenyl,        4-fluoro-3-(trifluoromethyl)phenyl, 4-fluoro-3-iodophenyl,        3-fluoro-4-iodophenyl, 3,4-di-fluorophenyl, 4-ethylphenyl,        3-fluoro-4-methylphenyl, 3-fluoro-4-ethylphenyl,        3-chloro-4-fluorophenyl, 3-fluoro-4-chlorophenyl,        2-methyl-3-methoxyphenyl, 2-ethyl-3-methoxyphenyl,        2-ethyl-3,4-ethylenedioxyphenyl, 3-nitrophenyl, 4-iodophenyl,        3-fluoro-4-trifluoromethylphenyl, 3-methylphenyl,        4-methylphenyl, 4-chlorophenyl, 3-trifluoromethylphenyl,        3-methoxyphenyl, 3-chloro-6-pyridyl, 2-chloro-4-pyridyl,        phenylamino, 3-chlorophenylamino, 3-methylphenylamino,        4-chlorophenylamino, and 4-methylphenylamino;    -   R² is hydrogen, methyl or CF₃;    -   R³ is hydrogen, methyl or CF₃;    -   R⁴ is hydrogen;    -   R⁵ is optionally substituted phenyl, wherein the substituents        are selected from the group consisting of cyano, nitro, halogen,        (C₁-C₃)alkyl, halo(C₁-C₃)alkyl, (C₁-C₃)alkoxy,        halo(C₁-C₃)alkoxy, (C₃)alkenyloxy, (C₃)alkynyloxy,        (C₁-C₃)alkylthio, halo(C₁-C₃)alkylthio, (C₁-C₃)alkylsulfinyl,        halo(C₁-C₃)alkylsulfinyl, (C₁-C₃)alkylsulfonyl,        halo(C₁-C₃)alkylsulfonyl, (C₁-C₃)alkoxy(C₁-C₃)alkyl,        (C₁-C₂)alkylthio(C₁-C₂)alkyl, and (C₁-C₃)alkoxycarbonyl;    -   R⁶ is selected from the group consisting of hydrogen, formyl,        (C₁-C₃)alkylcarbonyl, and cyclo(C₃-C₆)alkylcarbonyl; and    -   R⁷, R⁸, R⁹, R¹⁰ are independently selected from the group        consisting of hydrogen, cyano, nitro, chlorine, fluorine,        methyl, trifluoromethyl, difluoromethyl, methoxy,        trifluoromethoxy, difluoromethoxy, methylthio,        trifluoromethylthio, difluoromethylthio, methylsulfinyl,        trifluoromethylsulfinyl, difluoromethylsulfinyl, methylsulfonyl,        trifluoromethylsulfonyl, difluoromethylsulfonyl, methoxymethyl,        and methoxycarbonyl, or R⁷/R⁸, R⁸/R⁹, or R⁹/R¹⁰ form a 5- or        6-membered heterocyclic ring.

In another embodiment, the activating ligand is a compound havingFormula IV wherein Q is O, R² is methyl, and R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ are defined according to Table 4.

TABLE 4 Ligand Components R³, R⁴, R⁷, R⁹, R¹ and R¹⁰ R⁵ R⁶ R⁸ Stereo.¹n-Hexyl H Ph H H cis n-Heptyl H Ph H H cis n-Bu H Ph H H cis3-CF₃-4-F—Ph H 4-F—Ph H F trans 3-CF₃-4-F—Ph H 4-F—Ph H F cis3-Cl-4-F—Ph H 4-F—Ph H F trans 3-Cl-4-F—Ph H 4-F—Ph H F cis 4-F—Ph H4-F—Ph H F trans 4-F—Ph H 4-F—Ph H F cis Ph H 4-F—Ph H F trans Ph H4-F—Ph H F cis 3-F-4-Me—Ph H 4-F—Ph H F cis 3-Me-4-F—Ph H 4-F—Ph H F cis3-F-4-Me—Ph H 4-F—Ph H F trans 3,4-di-F—Ph H Ph H H cis 3-F-4-Me—Ph H PhH H cis 3-F-4-CF₃—Ph H Ph H H cis 3,4-di-F—Ph H Ph H H trans 3-F-4-Me—PhH Ph H H trans 3-F-4-CF₃—Ph H Ph H H trans 3,4-di-F—Ph H 4-Me—Ph H Mecis 3-F-4-Me—Ph H 4-Me—Ph H Me cis 3-F-4-CF₃—Ph H 4-Me—Ph H Me cis3,4-di-F—Ph H 4-F—Ph H F trans 3-F-4-CF₃—Ph H 4-F—Ph H F trans3,4-di-F—Ph H 4-F—Ph H F cis 3-F-4-CF₃—Ph H 4-F—Ph H F cis 3-F-4-Me—Ph H4-Me—Ph H Me trans 4-Cl—Ph H Ph H H trans 4-CH₃OC(O)—Ph H Ph H H trans3,4-OCH₂O—Ph H Ph H H trans 4-Cl—Ph H 4-Me—Ph H Me trans 4-CH₃OC(O)—Ph H4-Me—Ph H Me trans 3,4-OCH₂O—Ph H 4-Me—Ph H Me trans 4-Cl—Ph H 4-F—Ph HF trans 4-Et—Ph H 4-F—Ph H F trans 4-CH₃OC(O)—Ph H 4-F—Ph H F trans3,4-OCH₂O—Ph H 4-F—Ph H F trans 4-Me—Ph H Ph H H 80:20 cis:trans 4-Me—PhH 4-F—Ph H H 75:25 cis:trans 4-Me—Ph H 2-Cl—Ph H H 80:20 cis:trans4-Me—Ph H 3-Cl—Ph H H 50:50 cis:trans 4-Me—Ph H 4-Cl—Ph H H 80:20cis:trans 4-Me—Ph H 3-Me—Ph H H 60:40 cis:trans 4-Me—Ph H 4-Me—Ph H H70:30 cis:trans 4-Me—Ph H 3-MeO—Ph H H 60:40 cis:trans 4-Me—Ph H4-MeO—Ph H H 80:20 cis:trans 3-F-4-Me—Ph H, 4-F—Ph H H 60:40 (R⁹ = Cl)cis:trans 3-F-4-CF₃—Ph H 4-Me—Ph H Me trans 2-Me-3-MeO—Ph H Ph H H cis2-F—Ph H 4-Me—Ph H Me cis 2-Me—Ph H 4-Me—Ph H Me cis 2-MeO—Ph H 4-Me—PhH Me cis 2-Me-3-MeO—Ph H 4-Me—Ph H Me cis 2-F—Ph H 4-F—Ph H F cis2-Me—Ph H 4-F—Ph H F cis 2-MeO—Ph H 4-F—Ph H F cis 2-Me-3-MeO—Ph H4-F—Ph H F cis 4-Et—Ph H Ph H H trans 4-Et—Ph H 4-Me—Ph H Me trans4-Cl—Ph H Ph H H cis 4-Et—Ph H Ph H H cis 4-Cl—Ph H 4-Me—Ph H Me cis4-Et—Ph H 4-Me—Ph H Me cis 4-Cl—Ph H 4-F—Ph H F cis 4-Et—Ph H 4-F—Ph H Fcis Ph H Ph H H cis 3-F—Ph H Ph H H cis 2-CF₃—Ph H Ph H H cis 3-CF₃—Ph HPh H H cis 4-CF₃—Ph H Ph H H cis Ph H 4-Me—Ph H Me cis 3-F—Ph H 4-Me—PhH Me cis 2-CF₃—Ph H 4-Me—Ph H Me cis 3-CF₃—Ph H 4-Me—Ph H Me cis4-CF₃—Ph H 4-Me—Ph H Me cis 3-F—Ph H 4-F—Ph H F cis 2-CF₃—Ph H 4-F—Ph HF cis 3-CF₃—Ph H 4-F—Ph H F cis 4-CF₃—Ph H 4-F—Ph H F cis 3-MeO—Ph H PhH H cis 4-Me—Ph H Ph H H cis 4-MeO—Ph H Ph H H cis 4-CH₃OC(O)—Ph H Ph HH cis 3-Me—Ph H 4-Me—Ph H Me cis 3-MeO—Ph H 4-Me—Ph H Me cis 4-Me—Ph H4-Me—Ph H Me cis 4-MeO—Ph H 4-Me—Ph H Me cis 4-CH₃OC(O)—Ph H 4-Me—Ph HMe cis 3-Me—Ph H 4-F—Ph H F cis 3-MeO—Ph H 4-F—Ph H F cis 4-Me—Ph H4-F—Ph H F cis 4-MeO—Ph H 4-F—Ph H F cis 4-CH₃OC(O)—Ph H 4-F—Ph H F cis4-MeO—Ph H Ph H H trans 4-Me—Ph H Ph H H trans Ph H Ph H H trans4-MeO—Ph H 4-Me—Ph H Me trans 4-Me—Ph H 4-Me—Ph H Me trans Ph H 4-Me—PhH Me trans 4-MeO—Ph H 4-F—Ph H F trans 4-Me—Ph H 4-F—Ph H F trans6-Cl-3-pyridyl H Ph H H cis 5-isoxazolyl H Ph H H cis 3-F-4-Cl—Ph H Ph HH cis 2-Cl-4-pyridyl H Ph H H cis 2-Et-3-MeO—Ph H Ph H H cis3-Cl-6-pyridyl H 4-Me—Ph H Me cis 5-isoxazolyl H 4-Me—Ph H Me cis3-F-4-Cl—Ph H 4-Me—Ph H Me cis 2-Cl-4-pyridyl H 4-Me—Ph H Me cis2-Et-3-MeO—Ph H 4-Me—Ph H Me cis 3-Cl-6-pyridyl H 4-F—Ph H F cis5-isoxazolyl H 4-F—Ph H F cis 3-F-4-Cl—Ph H 4-F—Ph H F cis2-Cl-4-pyridyl H 4-F—Ph H F cis 2-Et-3-MeO—Ph H 4-F—Ph H F cis 2-ThienylH Ph Ac H Styryl H Ph Ac H 4-Cl—Ph H Ph 4-MeO—Ph—C(O) H furan-2-ylvinylH Ph H H 2-Thienyl H Ph H H 4-t-butyl-Ph H Ph Ac H 4-F—Ph H 4-Me—Ph H MeBenzosuccinimidyl-methyl H 4-Me—Ph H Me n-Pr H 4-F—Ph benzoyl H n-OctylH Ph H H cis Me H Ph 4-F—Ph—C(O) H 2-Cl—PhOCH₂ H Ph H H Benzyl H Ph H H4-MeO—Ph H Ph 2-thiophenyl-C(O) H Me H Ph 4-Me—Ph—C(O) H 3-MeO—Ph H Phn-hexanoyl H 4-t-butyl-Ph H Ph H H cis 4-MeO—Ph H, 2-Me—Ph H H (R¹⁰ =Me) 3-F—Ph H Ph 3-F—Ph(CO) H Ph H 3-MeO—Ph H H 4-n-pentyl-Ph H Ph H H2-furanyl H Ph H H Ph H 3-MeO—Ph Ac H 4-Me—Ph H Ph 3-MeO—PhC(O) H Me HPh 3-MeO—PhC(O) H 4-Me—Ph H Ph 4-F—Ph—C(O) H 4-Cl—Ph H 4-Me—Ph H MeCO₂Et H Ph EtOC(O)C(O) H 3,4-di-MeO-styryl H Ph H H cis Styryl H Phstyryl-C(O) H 3-Br—Ph H Ph H H Ph H 4-Me—Ph Ac H 4-MeO-styryl H Ph Ac HBenzosuccinimidyl-methyl H Ph H H 4-MeO—Ph H 4-Me—Ph H Me trans 4-MeO—PhH Ph 4-MeO—Ph—C(O) H 3-NO₂—Ph H 4-Me—Ph H Me cyclopropyl H Phcyclopropyl-C(O) H Me H 3-MeO—Ph benzoyl H 4-n-propyl H, 2-Me—Ph H H(R¹⁰ = Me) 3-NO₂—Ph H Ph H H cis 4-F—PhOCH₂ H Ph H H n-Pr H Ph3-MeO—PhC(O) H 4-Cl—Ph H Ph 4-Me—Ph—C(O) H 4-Et—Ph H, 2-Me—Phstyryl-C(O) H (R¹⁰ = Me) Styryl H Ph H H cis 3-Me—Ph H Ph 3-Me—Ph—C(O) H3,4-di-Cl—Ph H Ph H H cis 3-OH—Ph H Ph 3-Br—Ph(CO) H succinimidylmethylH Ph H H 4-I—Ph H Ph H H cis 1-naphthylmethyl H Ph H H cyclohexylethyl HPh Ph H CO₂Et H Ph H H 4-F—Ph H Ph 4-F—Ph—C(O) H 4-n-propyl-Ph H Ph H H3-F—Ph H 4-Me—Ph H Me trans 4-CH₃S(O₂)NH—Ph H Ph H H NHPh H Ph H H4-MeO-styryl H Ph H H cis i-Pr H 4-NO₂—Ph benzoyl H 3-Cl-benzofuran-2-ylH Ph 3-Cl-benzothiophen- H 2-yl 4-Cl—PhOCH₂ H Ph H H 4-MeO—Ph H Ph4-MeO-styryl H CF₃ H Ph CF₃C(O) H Et H Ph 4-NO₂—Ph—C(O) H Ph H, 2-Me—PhH H cis (R¹⁰ = Me) Me H Ph 2-F—Ph—C(O) H n-pentyl Ph 2-F—Ph—C(O) H4-Me—Ph H, 2-Me—Ph H H cis (R¹⁰ = Me) 3-F-4-Me—Ph H 4-Me—Ph3-F-4-Me—Ph(CO) Me trans 3-F-4-CF₃—Ph H 4-Me—Ph 3-F-4-CF₃—Ph—C(O) Metrans 4-Cl—Ph H Ph 4-Cl—Ph—C(O) H trans 4-Et—Ph H Ph 4-Et—Ph—C(O) Htrans 4-Cl—Ph H 4-Me—Ph 4-Cl—Ph—C(O) Me trans 4-Et—Ph H 4-Me—Ph4-Et—Ph—C(O) Me trans 3,4-OCH₂O—Ph H 4-Me—Ph 3,4-OCH₂O—Ph—C(O) Me trans3-F-4-Me—Ph H 4-F—Ph Ac F trans 3-F-4-Me—Ph H 4-F—Ph 3-F-4-Me—Ph(CO) Ftrans 3-F-4-Me—Ph H 4-F—Ph 3-F-4-Me—Ph(CO) F cis 3-Me—Ph H Ph H H trans3-F—Ph H Ph H H trans 3-MeO—Ph H Ph H H trans 3-CF₃—Ph H Ph H H trans3-Me—Ph H 4-Me—Ph H Me trans 3-F—Ph H 4-Me—Ph H Me trans 3-MeO—Ph H4-Me—Ph H Me trans 3-CF₃—Ph H 4-Me—Ph H Me trans 3-Me—Ph H 4-F—Ph H Ftrans 3-F—Ph H 4-F—Ph H F trans 3-MeO—Ph H 4-F—Ph H F trans 3-CF₃—Ph H4-F—Ph H F trans NHEt H Ph H H cis NEPh H Ph H H cis 4-Cl—Ph—NH H Ph H Hcis 3-Cl—Ph—NH H Ph H H cis 4-Me—Ph—NH H Ph H H cis 3-Me—Ph—NH H Ph H Hcis NHPh H 4-Me—Ph H Me cis 4-Cl—Ph—NH H 4-Me—Ph H Me cis 3-Cl—Ph—NH H4-Me—Ph H Me cis 4-Me—Ph—NH H 4-Me—Ph H Me cis 3-Me—Ph—NH H 4-Me—Ph H Mecis NHPh H 4-F—Ph H F cis 4-Cl—Ph—NH H 4-F—Ph H F cis 3-Cl—Ph—NH H4-F—Ph H F cis 4-Me—Ph—NH H 4-F—Ph H F cis 3-Me—Ph—NH H 4-F—Ph H F cis¹Relative stereochemistry at 2- and 4-positions

In another embodiment, the activating ligand is a compound havingFormula V, VI, or VII:

wherein Q¹ and Q² are independently selected from the group consistingof O and S;

-   -   n=1 or 2;    -   R¹ is selected from the group consisting of (C₁-C₆)alkyl,        (C₃-C₆)cycloalkyl, (C₁-C₆)haloalkyl, (C₃-C₆)halocycloalkyl,        (C₂-C₆)alkenyl, (C₂-C₆)haloalkenyl, (C₂-C₆)alkynyl,        (C₂-C₆)haloalkynyl, (C₁-C₆)alkoxy, (C₃-C₆)cycloalkoxy,        (C₁-C₆)haloalkoxy, (C₃-C₆)halocycloalkoxy, (C₂-C₆)alkenyloxy,        (C₂-C₆)alkynyloxy, (C₁-C₆)alkylthio, (C₃-C₆)cycloalkylthio,        (C₁-C₆)haloalkylthio, (C₃-C₆)halocycloalkylthio,        (C₁-C₆)alkylamino, (C₃-C₆)cycloalkylamino,        (C₁-C₆)haloalkylamino, (C₃-C₆)halocycloalkylamino,        di(C₁-C₆)alkylamino, di(C₃-C₆)cycloalkylamino,        di(C₁-C₆)haloalkylamino, di(C₃-C₆)halocycloalkylamino,        (C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)althylthio(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfinyl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₆)alkylamino(C₁-C₆)alkyl,        (C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl, optionally        substituted phenyl, optionally substituted 1-naphthyl,        optionally substituted 2-naphthyl, optionally substituted        phenyl(C₁-C₃)alkyl, optionally substituted phenyl(C₂-C₃)alkenyl,        optionally substituted naphthyl(C₁-C₃)alkyl, optionally        substituted phenoxy(C₁-C₃)alkyl, optionally substituted        phenylamino, and optionally substituted heterocycle;    -   R² and R³ are independently selected from the group consisting        of hydrogen, cyano, aminocarbonyl, carboxy, (C₁-C₆)alkyl,        (C₃-C₆)cycloalkyl, halo(C₁-C₆)alkyl, (C₃-C₆)halocycloalkyl,        (C₂-C₆)alkenyl, (C₃-C₆)cycloalkenyl, (C₂-C₆)haloalkenyl,        (C₂-C₆)alkynyl, (C₁-C₆)alkylsulfonyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,        (C₁-C₆)althylthio(C₁-C₆)alkyl, (C₁-C₆)alkylsulfinyl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₆)alkylamino(C₁-C₆)alkyl, (C₁-C₆)alkylcarbonyl,        (C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkylaminocarbonyl,        di(C₁-C₆)alkylaminocarbonyl,        (C₁-C₆)alkylaminocarbonyl(C₁-C₆)alkyl,        di(C₁-C₆)alkylaminocarbonyl(C₁-C₆)alkyl,        (C₁-C₆)alkylcarbonylamino(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonyl,        (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl,        hydroxy(C₁-C₆)alkyl, carboxy(C₁-C₆)alkyl, optionally substituted        phenyl, optionally substituted phenyl(C₁-C₆)alkyl, optionally        substituted benzoyl, optionally substituted naphthyl, optionally        substituted heterocycle, and optionally substituted        heterocyclylcarbonyl, or    -   R² and R³ are joined together with the carbon to which they are        attached to form an unsubstituted or substituted, partially        unsaturated or saturated optionally substituted 3-, 4-, 5-, 6-,        7- or 8-membered carbocyclic or heterocyclic ring, wherein the        heterocyclic ring contains from one to three heteroatoms        selected from O, N, or S;    -   R⁴ is selected from the group consisting of (C₁-C₆)alkyl,        (C₃-C₆)cycloalkyl, (C₁-C₆)haloalkyl, (C₃-C₆)halocycloalkyl,        (C₂-C₆)alkenyl, (C₂-C₆)haloalkenyl, (C₂-C₆)alkynyl,        (C₂-C₆)haloalkynyl, (C₁-C₆)alkoxy, (C₃-C₆)cycloalkoxy,        (C₁-C₆)haloalkoxy, (C₃-C₆)halocycloalkoxy, (C₂-C₆)alkenyloxy,        (C₂-C₆)alkynyloxy, (C₁-C₆)alkylthio, (C₃-C₆)cycloalkylthio,        (C₁-C₆)haloalkylthio, (C₃-C₆)halocycloalkylthio,        (C₁-C₆)alkylamino, (C₃-C₆)cycloalkylamino,        (C₁-C₆)haloalkylamino, (C₃-C₆)halocycloalkylamino,        di(C₁-C₆)alkylamino, di(C₃-C₆)cycloalkylamino,        di(C₁-C₆)haloalkylamino, di(C₃-C₆)halocycloalkylamino,        (C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)althylthio(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfinyl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₆)alkylamino(C₁-C₆)alkyl,        (C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl, optionally        substituted phenyl, optionally substituted 1-naphthyl,        optionally substituted 2-naphthyl, optionally substituted        phenyl(C₁-C₃)alkyl, optionally substituted phenyl(C₂-C₃)alkenyl,        optionally substituted naphthyl(C₁-C₃)alkyl, optionally        substituted phenoxy(C₁-C₃)alkyl, optionally substituted        phenylamino, and optionally substituted heterocycle;    -   R⁵ is selected from the group consisting of (C₁-C₆)alkyl,        (C₃-C₆)cycloalkyl, (C₁-C₆)haloalkyl, (C₃-C₆)halocycloalkyl,        (C₂-C₆)alkenyl, (C₂-C₆)haloalkenyl, (C₂-C₆)alkynyl,        (C₂-C₆)haloalkynyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,        (C₁-C₆)althylthio(C₁-C₆)alkyl, (C₁-C₆)alkylsulfinyl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₆)alkylamino(C₁-C₆)alkyl,        (C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl, optionally        substituted phenyl, optionally substituted 1-naphthyl,        optionally substituted 2-naphthyl, optionally substituted        phenyl(C₁-C₃)alkyl, optionally substituted phenyl(C₂-C₃)alkenyl,        optionally substituted naphthyl(C₁-C₃)alkyl, optionally        substituted phenoxy(C₁-C₃)alkyl, optionally substituted        phenylamino, and optionally substituted heterocycle; and    -   R⁶ and R⁷ are independently selected from the group consisting        of (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, (C₁-C₆)haloalkyl,        (C₃-C₆)halocycloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)haloalkenyl,        (C₂-C₆)alkynyl, (C₂-C₆)haloalkynyl, (C₁-C₆)alkoxy,        (C₃-C₆)cycloalkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)halocycloalkoxy,        (C₂-C₆)alkenyloxy, (C₂-C₆)alkynyloxy, (C₁-C₆)alkylthio,        (C₃-C₆)cycloalkylthio, (C₁-C₆)haloalkylthio,        (C₃-C₆)halocycloalkylthio, (C₁-C₆)alkylamino,        (C₃-C₆)cycloalkylamino, (C₁-C₆)haloalkylamino,        (C₃-C₆)halocycloalkylamino, di(C₁-C₆)alkylamino,        di(C₃-C₆)cycloalkylamino, di(C₁-C₆)haloalkylamino,        di(C₃-C₆)halocycloalkylamino, (C₁-C₆)alkoxy(C₁-C₆)alkyl,        (C₁-C₆)althylthio(C₁-C₆)alkyl, (C₁-C₆)alkylsulfinyl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,        di(C₁-C₆)alkylamino(C₁-C₆)alkyl,        (C₁-C₆)alkylcarbonyl(C₁-C₆)alkyl, cyano(C₁-C₆)alkyl, optionally        substituted phenyl, optionally substituted phenyl(C₁-C₆)alkyl,        optionally substituted heterocycle, optionally substituted        phenoxy, optionally substituted heterocycloxy, optionally        substituted phenylthio, optionally substituted heterocyclylthio,        optionally substituted naphthyl, optionally substituted        phenylamino, optionally substituted heterocyclylamino,        optionally substituted N-phenyl-N—(C₁-C₆)alkylamino, and        optionally substituted N-heterocyclyl-N—(C₁-C₆)alkylamino.

In another embodiment, the activating ligand is a compound havingFormula V, wherein:

-   -   Q¹ is O;    -   R¹ is substituted phenyl wherein the substituents are        independently selected from the group consisting of (C₁-C₂)alkyl        and (C₁-C₂)alkoxy; or two adjacent positions are joined together        with the atoms to which they are attached to form an        unsubstituted or substituted, unsaturated, partially        unsaturated, or saturated 5-, 6- or 7-membered carbocyclic or        heterocyclic ring, wherein the heterocyclic ring contains from        one to two oxygen atoms and one to four substituents are        independently selected from the group consisting of: cyano,        (C₁-C₂)alkyl, (C₁-C₂)alkylamino, di(C₁-C₂)alkylamino,        (C₁-C₂)alkoxycarbonyl, (C₁-C₂)alkylaminocarbonyl,        di(C₁-C₂)alkylaminocarbonyl, oxo, and methoxyimino;    -   R² and R³ are independently selected from the group consisting        of (C₁-C₆)alkyl, (C₃-C₆)cycloalkyl, halo(C₁-C₆)alkyl,        (C₁-C₃)alkoxy(C₁-C₃)alkyl, (C₁-C₃)althylthio(C₁-C₃)alkyl,        (C₁-C₃)alkylsulfinyl(C₁-C₃)alkyl,        (C₁-C₃)alkylsulfonyl(C₁-C₃)alkyl, (C₁-C₃)alkylamino(C₁-C₃)alkyl,        di(C₁-C₃)alkylamino(C₁-C₃)alkyl, (C₁-C₆)alkylcarbonyl,        (C₁-C₃)alkylcarbonyl(C₁-C₃)alkyl, (C₁-C₆)alkylaminocarbonyl,        di(C₁-C₆)alkylaminocarbonyl,        (C₁-C₃)alkylaminocarbonyl(C₁-C₃)alkyl,        di(C₁-C₃)alkylaminocarbonyl(C₁-C₃)alkyl,        (C₁-C₃)alkylcarbonylamino(C₁-C₃)alkyl, (C₁-C₆)alkoxycarbonyl,        and C₁-C₃)alkoxycarbonyl(C₁-C₃)alkyl; or    -   R² and R³ may be joined together with the carbon to which they        are attached to form an unsubstituted or substituted, partially        unsaturated or saturated 5-, 6- or 7-membered carbocyclic or        heterocyclic ring, wherein the heterocyclic ring contains one        heteroatom selected from O or S; and one to four substituents        are independently selected from the group consisting of        (C₁-C₃)alkyl, (C₁-C₃)alkylamino, di(C₁-C₃)alkylamino,        (C₁-C₄)alkoxycarbonyl, (C₁-C₃)alkylaminocarbonyl, and        di(C₁-C₃)alkylaminocarbonyl; and    -   R⁴ is selected from optionally substituted phenyl or pyridyl        wherein the substituents are independently selected from the        group consisting of (C₁-C₃)alkyl and (C₁-C₃)alkoxy;

In another embodiment, the activating ligand is a compound havingFormula V, wherein Q is oxygen, and R¹, R², R³, and R⁴ are definedaccording to Table 5.

TABLE 5 Ligand Components R¹ R² R³ R⁴ 2-Me-3-MeO—Ph —(CH₂)₄— Ph2-Me-3-MeO—Ph —(CH₂)₄— 3-Me—Ph 4-Et—Ph —(CH₂)₄— Ph 2-Me-3-MeO—Ph—(CH₂)₅— 3-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₄— 3-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₃—3-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 3-Me—Ph 2-Me-3-MeO—Ph Bn Me 3-Me—Ph2-Me-3-MeO—Ph —(CH₂)₂— 3-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₄— 3,5-diMe—Ph2-Me-3-MeO—Ph —(CH₂)₅— 3,5-diMe—Ph 2-Me-3-MeO—Ph Bn Me 3,5-diMe—Ph2-Me-3-MeO—Ph —(CH₂)₂— 3,5-diMe—Ph 2-Me-3-MeO—Ph —(CH₂)₃— 3,5-diMe—Ph2-Me-3-MeO—Ph —(CH₂)₅— 4-Me—Ph 2-Me-3-MeO—Ph Bn Me 4-Me—Ph 2-Me-3-MeO—Ph—(CH₂)₄— 3-Me-4-F—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 3-Me-4-F—Ph 2-Me-3-MeO—Ph—(CH₂)₂— 3-Me-4-F—Ph 2-Me-3-MeO—Ph i-Pr Me 3,5-diMe—Ph 2-Et-3-MeO—Ph—(CH₂)₄— Ph 2-Et-3,6-OCH₂CH₂O—Ph —(CH₂)₄— Ph 2-Me-3,4-OCH₂O—Ph —(CH₂)₄—Ph 2-Me-3-MeO—Ph —(CH₂)₂— 4-Me—Ph 2-Me-3-MeO—Ph —CH₂CH₂OCH₂CH₂— 3-Me—Ph2-Me-3-MeO—Ph —CH₂CH₂SCH₂CH₂— 3-Me—Ph 2-Me-3-MeO—Ph —CH₂CH₂OCH₂CH₂—3,5-diMe—Ph 2-Me-3-MeO—Ph —CH₂CH₂SCH₂CH₂— 3,5-diMe—Ph 2-Me-3-MeO—Ph—CH₂CH₂C(OCH₂CH₂O)CH₂CH₂— 3,5-diMe—Ph 2-Me-3-MeO—Ph i-Pr Me 2-MeO—Ph2-Me-3-MeO—Ph i-Pr Me 3-Me—Ph 2-Me-3-MeO—Ph i-Pr Me 3-MeO—Ph2-Me-3-MeO—Ph i-Pr Me 4-Me—Ph 2-Me-3-MeO—Ph i-Pr Me Ph 2-Me-3-MeO—Ph—CH₂CH₂C(OCH₂CH₂O)CH₂CH₂— 3-Me—Ph 2-Me-3-MeO—Ph Et Et 2-Me—Ph2-Me-3-MeO—Ph Et Et 2-MeO—Ph 2-Me-3-MeO—Ph Et Et 4-F—Ph 2-Me-3-MeO—Ph—(CH₂)₄— 2-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₄— 2-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₄—4-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₄— 4-F—Ph 2-Me-3-MeO—Ph —(CH₂)₄—3,4-OCH₂O—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 2-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₅—2-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 4-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₅—3,4-OCH₂O—Ph 2-Me-3-MeO—Ph Et Et 3-Me—Ph 2-Me-3-MeO—Ph Et Et 3-MeO—Ph2-Me-3-MeO—Ph Et Et 3-Me-4-F—Ph 2-Me-3-MeO—Ph Et Et 3,5-diMe—Ph2-Me-3-MeO—Ph i-Bu Me 3-Me—Ph 2-Me-3-MeO—Ph i-Bu Me 3-MeO—Ph2-Me-3-MeO—Ph i-Bu Me 3-Me-4-F—Ph 2-Me-3-MeO—Ph i-Bu Me 3,5-diMe—Ph2-Me-3-MeO—Ph i-Pr Me 3-Me-4-F—Ph 2-Me-3-MeO—Ph Ph i-Pr 3-Me—Ph2-Me-3-MeO—Ph Et Et 4-MeO—Ph 2-Me-3-MeO—Ph Et Et 3,4-OCH2O—Ph2-Me-3-MeO—Ph —(CH₂)₅— 4-F—Ph 2-Me-3-MeO—Ph —CH₂CH₂C(=O)CH₂CH₂— 3-Me—Ph2-Me-3-MeO—Ph —CH₂CH₂S(=O)₂CH₂CH₂— 3,5-diMe—Ph 2-Me-3-MeO—Ph i-Pr Me2-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 2,6-diMeO-3-pyridyl 2-Me-3-MeO—Ph—(CH₂)₄— 3,5-diMeO-4-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 3,5-diMeO-4-Me—Ph2-Me-3-MeO—Ph —(CH₂)₄— 3-MeO-4,5-diF—Ph 2-Me-3-MeO—Ph —(CH₂)₅—3-MeO-4,5-diF—Ph 2-Me-3-MeO—Ph —(CH₂)₅— Ph 2-Me-3-MeO—Ph —(CH₂)₆—2-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₆— 3,5-diMe—Ph 2-Me-3-MeO—Ph 4-F—Ph Me2-MeO—Ph 2-Me-3-MeO—Ph 4-F—Ph Me 3,5-diMe—Ph 2-Me-3-MeO—Ph Me Me2-MeO—Ph 2-Me-3-MeO—Ph Me Me 3,5-diMe—Ph 2-Me-3-MeO—Ph Me Me Ph2-Me-3-MeO—Ph Et Et 4-Me—Ph 2-Me-3-MeO—Ph Et Et Ph 2-Me-3-MeO—Ph—(CH₂)₄— 4-Me—Ph 2-Et-3,4-OCH₂CH₂O—Ph —(CH₂)₅— 3,5-di-Me—Ph2-Me-3,4-OCH₂O—Ph —(CH₂)₅— 3,5-di-Me—Ph 3,4-OCH₂CH₂O—Ph —(CH₂)₅—3,5-di-Me—Ph 3,4-CH₂OCH₂O—Ph —(CH₂)₅— 3,5-di-Me—Ph 2-Et-3,4-OCH₃CH₂O—Ph—(CH₂)₄— 3,5-di-Me—Ph 2-Me-3,4-OCH₂O—Ph —(CH₂)₄— 3,5-di-Me—Ph3,4-OCH₂CH₂O—Ph —(CH₂)₄— 3,5-di-Me—Ph 3,4-CH₂OCH₂O—Ph —(CH₂)₄—3,5-di-Me—Ph 3,4-OCH₂O—Ph —(CH₂)₄— 3,5-di-Me—Ph 2-Me—Ph —(CH₂)₄—3,5-di-Me—Ph Ph t-Bu H 4-Cl—Ph 4-Cl—Ph —(CH₂)₄— Ph Me Ph H 4-Me—Ph Me4-Me—Ph H Ph Me Ph H Ph 4-Cl—Ph Me Me Ph 4-Me—Ph t-Bu H Ph 2,3-di-Me—Pht-Bu H Ph 4-NO₂—Ph t-Bu H Ph 2-Me-3-MeO—Ph —(CH₂)₂— 3-MeO—Ph2-Me-3-MeO—Ph Benzyl Me 3-MeO—Ph 2-Me-3-MeO—Ph —(CH₂)₂— 2-Me—Ph3-Me-benzofuran-2-yl —(CH₂)₄— Ph Ph Me Me Ph 2-Me—Ph Me Me Ph3,4-OCH₂O—Ph Me Me Ph 3-MeO—Ph Me Me Ph 4-Et—Ph Me Me Ph 2-Me-3-MeO—Ph—CH₂CH₂N(C(O)OtBu)CH₂CH₂— 3-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂N(C(O)OtBu)CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph i-Pr Me3,4-OCH₂O—Ph 2-Me-3-MeO—Ph i-Pr Me Me 2-Me-3-MeO—Ph t-Bu H 3-Me—Ph2-Me-3-MeO—Ph t-Bu H 3-MeO—Ph 2-Me-3-MeO—Ph t-Bu H 3,5-di-Me—Ph 2-MeO—PhMe Me 3-Me—Ph 2-MeO—Ph Me Me 3-MeO—Ph 2-Me-3-MeO—Ph i-Bu Me 4-MeO—Ph2-MeO—Ph Me Me 3,5-di-Me—Ph 2-Me-3-MeO—Ph (CH₂)₅ n-Bu Ph Me Me Et3-MeO—Ph Me Me Et 3,4-OCH₂O—Ph Me Me Et 2-Me—Ph Me Me Et 4-Et—Ph Me MeEt Ph Me Me 3,5-di-Me—Ph 2-Me—Ph Me Me 3,5-di-Me—Ph 3-MeO—Ph Me Me3,5-di-Me—Ph 4-Et—Ph Me Me 3,5-di-Me—Ph 3,4-OCH₂O—Ph Me Me 3,5-di-Me—PhPh —(CH₂)₄— Et 2-Me—Ph —(CH₂)₄— Et 3-MeO—Ph —(CH₂)₄— Et 4-Et—Ph —(CH₂)₄—Et 3,4-OCH₂O—Ph —(CH₂)₄— Et Ph —(CH₂)₄— 3,5-di-Me—Ph 3-MeO—Ph —(CH₂)₄—3,5-di-Me—Ph 4-Et—Ph —(CH₂)₄— 3,5-di-Me—Ph Ph —(CH₂)₄— Ph 2-Me—Ph—(CH₂)₄— Ph 3-MeO—Ph —(CH₂)₄— Ph 3,4-OCH₂O—Ph —(CH₂)₄— Ph 2-Et-3-MeO—Ph—(CH₂)₅— 3,5-di-Me—Ph 2-Et-3-MeO—Ph —(CH₂)₄— 3,5-di-Me—Ph CF₃ —(CH₂)₄—3,5-di-Me—Ph 2-Me-3-MeO—Ph —CH₂N[(C═O)Ot-Bu]CH₂CH₂CH₂— 3,5-di-Me—Ph2-Me-3-MeO—Ph —CH₂CH₂NHCH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂NHCH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph —CH₂CH₂N[(C═O)CH₃]CH₂CH₂—3,5-di-Me—Ph 2-Me-3-MeO—Ph —CH₂CH₂N[(C═O)(C═O)OEt]CH₂CH₂— 3,5-di-Me—Ph2-Me-3-MeO—Ph —CH₂CH₂N[S(O)₂CH₃]CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂N[CH₂(C═O)OEt]CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂N[(C═O)CH₃]CH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂N[(C═O)(C═O)OEt]CH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂N[S(O)₂CH₃]CH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂N[CH₂(C═O)OCH₃]CH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂N[(C═O)NHEt]CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂N[(C═O)OiPr]CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂N[CH₂CN]CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂N[(C═O)NHEt]CH₂CH₂CH₂— 3,5-di-Me—Ph 2-Me-3-MeO—Ph—CH₂CH₂CH₂N(CH₃)CH₂— 3,5-di-Me—Ph 2-NH₂—Ph Et H Ph 4-Et—Ph —(CH₂)₅—3,5-di-Cl—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 2-MeO-5-F—Ph 2-Me-3-MeO—Ph —(CH₂)₅—2-MeO-5-Me—Ph 2-Me-3-MeO—Ph —(CH₂)₅— 2,5-di-MeO—Ph 2-Me-3-MeO—Ph—(CH₂)₅— 4-Me-2-pyridyl 2-Me-3-MeO—Ph —(CH₂)₅— 6-Me-2-pyridyl 4-Et—Ph—(CH₂)₅— 2-MeO-5-F—Ph 4-Et—Ph —(CH₂)₅— 2-MeO-5-Me—Ph 4-Et—Ph —(CH₂)₅—2,5-di-MeO—Ph 4-Et—Ph —(CH₂)₅— 4-Me-2-pyridyl 4-Et—Ph —(CH₂)₅—6-Me-2-pyridyl 4-Et—Ph —(CH₂)₅— 2-MeO—Ph 4-Et—Ph —(CH₂)₅— 3,5-di-Me—Ph4-Et—Ph —(CH₂)₅— 3-Me—Ph 2-Me-3-MeO—Ph i-Pr Et 2-MeO—Ph 2-Me-3-MeO—Phi-Pr Et 3,5-di-Me—Ph

In another embodiment, the activating ligand is a compound havingFormula VI, wherein n is 2, and R², R³, R⁴, and R⁵ are defined accordingto Table 6.

TABLE 6 Ligand Components R²/R³ R⁴ R⁵ —(CH₂)₅— 3,5-di-Me—Ph4H-benzo[1,3]dioxine-6-yl —(CH₂)₄— 3,5-di-Me—Ph 4-Me—Ph —(CH₂)₅—3,5-di-Cl—Ph 4-Me—Ph —(CH₂)₅— 3,5-di-Cl—Ph 3-MeO—Ph

In another embodiment, the activating ligand is a compound havingFormula VIII:

wherein:

-   -   X and X′ are independently O or S;    -   R¹ is selected from the group consisting of hydrogen,        (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl,        (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, benzyloxy,        optionally substituted phenyl, optionally substituted naphthyl        wherein the substituents are independently 1 to 3 halo, nitro,        (C₁-C₆) alkoxy, (C₁-C₆)alkyl, or amino, optionally substituted        benzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl, or        benzofuran-3-yl wherein the substituents are independently 1 to        3 halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or        (C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), optionally substituted 2-,        3-, or 4-pyridyl wherein the substituents are independently 1 to        3 halo, cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or        (C₁-C₆)haloalkoxy, optionally substituted 5-membered heterocycle        selected from furyl, thiophenyl, triazolyl, pyrrolyl,        isopyrrolyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl,        oxazolyl, or isooxazolyl wherein the substituents are        independently 1 to 3 halo, nitro, hydroxy, (C₁-C₆)alkyl,        (C₁-C₆)alkoxy, carboxy, (C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or        unsubstituted or substituted phenyl wherein the substituents are        independently 1 to 3 halo, nitro, (C₁-C₆)alkyl,        (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, carboxy,        (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)),        aromatic substituted or unsubstituted phenyl(C₁-C₆)alkyl,        phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein        the aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆) alkoxy, (C₁-C₆)alkyl, or amino, and aromatic substituted        or unsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, or        phenylcarbonylamino wherein the aromatic substituents are        independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl,        or amino;    -   wherein R^(a), R^(b), and R^(c) are independently H,        (C₁-C₆)alkyl, or phenyl;    -   R² and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,        (C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl,        (C₁-C₆)alkoxy(C₁-C₆)alkyl, phenyl, or together as an alkane        linkage (—(CH₂)_(x)—), an alkyloxylalkyl linkage        (—(CH₂)_(y)O(CH₂)_(z)—), an alkylaminoalkyl linkage        (—(CH₂)_(y)NR^(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage        (—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon        atom to which they are attached, wherein x=3 to 7, y=1 to 3, z=1        to 3, and R^(a) is H, (C₁-C₆)alkyl, or phenyl; and    -   R⁴ is optionally substituted phenyl, wherein the substituents        are independently 1 to 5 H; halo; nitro; cyano; hydroxy; amino        (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;        (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;        (C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;        (C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;        (C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)        alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted        with halo or (C₁-C₄)alkyl; formyl; carboxy;        (C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl;        (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido (—CONR^(a)R^(b));        amido (—NR^(a)COR^(b)); alkoxycarbonylamino (—NR^(a)CO₂R^(b));        alkylaminocarbonylamino (—NR^(a)CONR^(b)R); mercapto;        (C₁-C₆)alkylthio; (C₁-C₆) alkylsulfonyl; (C₁-C₆)alkylsulfoxido        (—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or optionally        substituted phenyl wherein the substituents are independently 1        to 3 halo, nitro, (C₁-C₆) alkoxy, (C₁-C₆)alkyl, or amino; or        when two adjacent positions on the phenyl ring are substituted        with alkoxy groups, these groups, together with the carbon atoms        to which they are attached, may be joined to form a 5- or        6-membered dioxolano (—OCH₂O—) or dioxano (—OCH₂CH₂O—)        heterocyclic ring; wherein R^(a), R^(b), and R^(c) are        independently H, (C₁-C₆)alkyl, or phenyl.

In another embodiment, the activating ligand is a compound havingFormula VIII, wherein:

-   -   X and X′ are O;    -   R¹ is phenyl, 4-chlorophenyl-, 4-ethylphenyl-,        2-ethyl-3,4-ethylenedioxyphenyl, 3-fluorophenyl-,        2-fluoro-4-ethylphenyl-, 2-methyl-3-methoxyphenyl-,        2-ethyl-3-methoxyphenyl, 3-methylphenyl-, 2-methoxyphenyl-,        2-nitrophenyl-, 3-nitrophenyl-, 2-furanyl-, benzyl-,        benzothiophene-2-yl-, phenylamino-, benzyloxymethyl,        phenoxymethyl-, 3-toluoylamino-, benzylamino-, benzoylamino-,        ethoxycarbonylethyl-, or 3-chloro-2,2,3,3-tetrafluoroethyl;    -   R² and R³ are independently methyl, ethyl, or together as a        tetramethylene (—(CH2)₄-), 4-pyrano (—CH₂CH₂OCH₂CH₂—), or        methylenebenzoethylene (—CH₂-1-benzo-2-CH₂CH₂—) linkage form a        ring with the carbon atom to which they are attached; and    -   R⁴ is phenyl, 4-biphenyl, 4-chlorophenyl, 2,4-dimethoxyphenyl,        3,5-dimethylphenyl, 2-methoxyphenyl, 3,4-methylenedioxyphenyl,        3-trifluoromethylphenyl, or 4-trifluromethoxyphenyl;

In another embodiment, the activating ligand is a compound havingFormula VIII selected from the group consisting of:

-   1-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-fluoro-benzamide;-   Furan-2-carboxylic acid    [3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-amide;-   3-Chloro-N-[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2,2,3,3-tetrafluoro-propionamide;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-4-ethyl-benzamide;-   2-Benzyloxy-N-[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-2-ethyl-3-methoxy-benzamide;-   2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-benzamide;-   Furan-2-carboxylic acid    [3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;-   2-Phenoxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-acetamide;-   N-(3-Phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-succinamic acid    ethyl ester;-   N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-benzamide;-   2-Ethyl-3-methoxy-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   1-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-3-phenyl-urea;-   2-Benzyloxy-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-benzamide;-   N-(3-Biphenyl-4-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-ethyl-3-methoxy-benzamide;-   N-[5,    5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   N-[5,    5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-ethyl-3-methoxy-benzamide;-   4-Chloro-N-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   1-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   4-Ethyl-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   1-Phenyl-3-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-urea;-   N-[5,    5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   2-Phenyl-N-(3-phenyl-1-oxa-2,4-di    aza-spiro[4.4]non-2-en-4-yl)-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-succinamic    acid ethyl ester;-   N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-benzamide;-   2-Benzyloxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-acetamide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.5]-7,8-benzo-dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamic    acid ethyl ester;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-4-ethyl-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-2-phenoxy-acetamide;-   N-(5,    5-Dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-methoxy-2-methyl-benzamide;-   N-(3-Phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-methoxy-2-methyl-benzamide,-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-2-phenyl-acetamide;-   Benzo[b]thiophene-2-carboxylic acid    [3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-2-phenoxy-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-2-ethyl-3-methoxy-benzamide;-   2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-acetamide;-   1-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-acetamide;-   1-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-3-phenyl-urea;-   N-[5,    5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   1-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-m-tolyl-urea;-   N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;-   3-Chloro-N-[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2,2,3,3-tetrafluoro-propionamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-benzamide;-   3-Chloro-2,2,3,3-tetrafluoro-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-propionamide;-   3-Chloro-2,2,3,3-tetrafluoro-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-propionamide;-   2-Benzyloxy-N-[5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-acetamide;-   1-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;-   Furan-2-carboxylic acid    [5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;-   Furan-2-carboxylic acid    (3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-amide;-   1-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   3-Chloro-N-[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2,2,    3,3-tetrafluoro-propionamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-methoxy-benzamide;-   2-Ethyl-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-methoxy-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]1-3-methyl-benzamide;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-phenyl-acetamide;-   Furan-2-carboxylic acid    [3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-succinamic    acid ethyl ester;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-2-phenyl-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide;-   Benzo[b]thiophene-2-carboxylic acid    [3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;-   1-Benzyl-3-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-urea;-   N-(3-Phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-benzamide;-   3-Chloro-N-[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-2,2,3,3-tetrafluoro-propionamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-nitro-benzamide;-   2-Ethyl-3-methoxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-benzamide;-   N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide,-   Furan-2-carboxyli c acid    [5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;-   1-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-phenyl-urea;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-nitro-benzamide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;-   Furan-2-carboxylic acid    (5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide;-   Furan-2-carboxylic acid    [3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]1-amide;-   N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-2-phenoxy-acetamide;-   Furan-2-carboxylic acid    [3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-amide;-   Benzo[b]thiophene-2-carboxylic acid    [5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;-   Benzo[b]thiophene-2-carboxylic acid    [5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;-   2-Benzyloxy-N-[3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;-   1-Benzoyl-3-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-urea;-   1-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-3-phenyl-urea;-   1-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   N-(5,5-Dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-benzamide;-   2-Benzyloxy-N-[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;-   N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   1-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;-   4-Ethyl-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-benzamide;-   4-Ethyl-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-succinamic    acid ethyl ester;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-phenoxy-acetamide;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   Benzo[b]thiophene-2-carboxylic acid    [3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;-   2-Phenyl-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-acetamide;-   1-Phenyl-3-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-urea;-   Benzo[b]thiophene-2-carboxylic acid    (5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide;-   N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   4-Ethyl-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-benzamide;-   Furan-2-carboxylic acid    [3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-amide;-   Benzo[b]thiophene-2-carboxylic acid    (3-benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-amide;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   N-(3-Biphenyl-4-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-benzamide;-   N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamic    acid ethyl ester;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-benzyloxy-acetamide;-   N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-2-phenyl-acetamide;-   N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;-   N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;-   Furan-2-carboxylic acid    (3-benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-amide;-   Benzo[b]thiophene-2-carboxylic acid    (3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-amide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide,-   Benzo[b]thiophene-2-carboxylic acid    [3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-amide;-   N-[5,    5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-succinamic    acid ethyl ester;-   2-Benzyloxy-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-acetamide;-   2-Benzyloxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-acetamide;-   N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-benzamide;-   N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   2-Phenoxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-acetamide;-   2-Ethyl-3-methoxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl)-benzamide;-   N-[5,    5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;-   Benzo[b]thiophene-2-carboxylic acid    [3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-amide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   N-[5,    5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;-   N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamic    acid ethyl ester;-   N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-2-fluoro-benzamide;-   4-Ethyl-2-fluoro-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-benzamide;-   N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl]-4-ethyl-2-fluoro-benzamide;-   N-(5,    5-Dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-2-fluoro-benzamide;-   5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid    (5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide; and-   5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid    [3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-amide.

In another embodiment, the activating ligand is a compound havingFormula IX or X:

wherein R¹, R², R³, and R⁴ are each independently:

-   -   a) H, (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl;        (C₁-C₆)hydroxyalkyl; (C₁-C₄)alkoxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl        optionally substituted with halo, cyano, hydroxyl, or        (C₁-C₄)alkyl; (C₂-C₆)alkynyl optionally substituted with halo,        cyano, hydroxyl, or (C₁-C₄)alkyl; (C₃-C₅)cycloalkyl optionally        substituted with halo, cyano, hydroxyl, or (C₁-C₄)alkyl;        oxiranyl optionally substituted with halo, cyano, or        (C₁-C₄)alkyl; or    -   b) unsubstituted or substituted benzyl wherein the substituents        are independently 1 to 5 H, halo, nitro, cyano, hydroxyl,        (C₁-C₆)alkyl, or (C₁-C₆)alkoxy; and    -   R⁵ is H; OH; F; Cl; or (C₁-C₆)alkoxy.

In another embodiment, the activating ligand is a compound selected fromthe group consisting of 20-hydroxyecdysone-2-methyl ether;20-hydroxyecdysone-3-methyl ether; 20-hydroxyecdysone-14-methyl ether;20-hydroxyecdysone-2,22-dimethyl ether; 20-hydroxyecdysone-3,22-dimethylether; 20-hydroxyecdysone-14,22-dimethyl ether;20-hydroxyecdysone-22,25-dimethyl ether;20-hydroxyecdysone-2,3,14,22-tetramethyl ether;20-hydroxyecdysone-22-n-propyl ether; 20-hydroxyecdysone-22-n-butylether; 20-hydroxyecdysone-22-allyl ether; 20-hydroxyecdysone-22-benzylether; 20-hydroxyecdysone-22-(28R,S)-2′-ethyloxiranyl ether; ponasteroneA-2-methyl ether; ponasterone A-14-methyl ether; ponasterone A-22-methylether; ponasterone A-2,22-dimethyl ether; ponasterone A-3,22-dimethylether; ponasterone A-14,22-dimethyl ether; dacryhainansterone-22-methylether; 25,26-didehydroponasterone A (iso-stachysterone C (Δ25(26)));shidasterone (stachysterone D); stachysterone C;22-deoxy-20-hydroxyecdysone (taxisterone); ponasterone A;polyporusterone B; 22-dehydro-20-hydroxyecdysone; 20-hydroxyecdysone;pterosterone; (25R)-inokosterone; (25S)-inokosterone; pinnatasterone;25-fluoroponasterone A; 24(28)-dehydromakisterone A; 24-epi-makisteroneA; makisterone A; 20-hydroxyecdysone-22-methyl ether;20-hydroxyecdysone-25-methyl ether; abutasterone;22,23-di-epi-geradiasterone; 20,26-dihydroxyecdysone (podecdysone C);24-epi-abutasterone; geradiasterone; 29-norcyasterone; ajugasterone B;24(28)[Z]-dehydroamarasterone B; amarasterone A; makisterone C;rapisterone C; 20-hydroxyecdysone-22,25-dimethyl ether;20-hydroxyecdysone-22-ethyl ether; carthamosterone;24(25)-dehydroprecyasterone; leuzeasterone; cyasterone;20-hydroxyecdysone-22-allyl ether;24(28)[Z]-dehydro-29-hydroxymakisterone C;20-hydroxyecdysone-22-acetate; viticosterone E (20-hydroxyecdysone25-acetate); 20-hydroxyecdysone-22-n-propyl ether; 24-hydroxycyasterone;ponasterone A 22-hemisuccinate; 22-acetoacetyl-20-hydroxyecdysone;canescensterone; 20-hydroxyecdysone-22-hemisuccinate;inokosterone-26-hemisuccinate; 20-hydroxyecdysone-22-benzoate;20-hydroxyecdysone-22-β-D-glucopyranoside;20-hydroxyecdysone-25-β-D-glucopyranoside; sileneoside A(20-hydroxyecdysone-22α-galactoside); 3-deoxy-1β,20-dihydroxyecdysone(3-deoxyintegristerone A); 2-deoxyintegristerone A; 1-epi-integristeroneA; integristerone A; sileneoside C (integristerone A 22α-galactoside);2,22-dideoxy-20-hydroxyecdysone; 2-deoxy-20-hydroxyecdysone;2-deoxy-20-hydroxyecdysone-3-acetate; 2-deoxy-20,26-dihydroxyecdysone;2-deoxy-20-hydroxyecdysone-22-acetate;2-deoxy-20-hydroxyecdysone-3,22-diacetate;2-deoxy-20-hydroxyecdysone-22-benzoate; ponasterone A 2-hemisuccinate;20-hydroxyecdysone-2-acetate; 20-hydroxyecdysone-2-hemisuccinate;20-hydroxyecdysone-2-β-D-glucopyranoside; 2-dansyl-20-hydroxyecdysone;20-hydroxyecdysone-2,22-dimethyl ether; ponasterone A3β-D-xylopyranoside (limnantheoside B); 20-hydroxyecdysone-3-methylether; 20-hydroxyecdysone-3-acetate;20-hydroxyecdysone-3β-D-xylopyranoside (limnantheoside A);20-hydroxyecdysone-3-β-D-glucopyranoside; sileneoside D(20-hydroxyecdysone-3α-galactoside); 20-hydroxyecdysone3β-D-glucopyranosyl-[1-3]-β-D-xylopyranoside (limnantheoside C);cyasterone-3-acetate; 2-dehydro-3-epi-20-hydroxyecdysone;3-epi-20-hydroxyecdysone (coronatasterone); rapisterone D;3-dehydro-20-hydroxyecdysone; 5β-hydroxy-25,26-didehydroponasterone A;5β-hydroxystachysterone C; 25-deoxypolypodine B; polypodine B;25-fluoropolypodine B; 5β-hydroxyabutasterone; 26-hydroxypolypodine B;29-norsengosterone, sengosterone; 6β-hydroxy-20-hydroxyecdysone;6α-hydroxy-20-hydroxyecdysone; 20-hydroxyecdysone-6-oxime; ponasterone A6-carboxymethyloxime; 20-hydroxyecdysone-6-carboxymethyloxime;ajugasterone C; rapisterone B; muristerone A; atrotosterone B;atrotosterone A; turkesterone-2-acetate; punisterone (rhapontisterone);turkesterone; atrotosterone C; 25-hydroxyatrotosterone B;25-hydroxyatrotosterone A; paxillosterone; turkesterone-2,22-diacetate;turkesterone-22-acetate; turkesterone-11α-acetate;turkesterone-2,11α-diacetate; turkesterone-11α-propionate;turkesterone-11α-butanoate; turkesterone-11α-hexanoate;turkesterone-11α-decanoate; turkesterone-11α-laurate;turkesterone-11α-myristate; turkesterone-11α-arachidate,22-dehydro-12β-hydroxynorsengosterone; 22-dehydro-12β-hydroxycyasterone;22-dehydro-12β-hydroxysengosterone; 14-deoxy(14α-H)-20-hydroxyecdysone;20-hydroxyecdysone-14-methyl ether; 14α-perhydroxy-20-hydroxyecdysone;20-hydroxyecdysone-2,3,14,22-tetramethyl ether;(20S)-22-deoxy-20,21-dihydroxyecdysone; 22,25-dideoxyecdysone;(22S)-20-(2,2′-dimethylfuranyl)ecdysone;(22R)-20-(2,2′-dimethylfuranyl)ecdysone; 22-deoxyecdysone25-deoxyecdysone; 22-dehydroecdysone; ecdysone; 22-epi-ecdysone;24-methylecdysone (20-deoxymakisterone A); ecdysone-22-hemisuccinate;25-deoxyecdysone-22-β-D-glucopyranoside; ecdysone-22-myristate;22-dehydro-20-iso-ecdysone; 20-iso-ecdysone; 20-iso-22-epi-ecdysone;2-deoxyecdysone; sileneoside E (2-deoxyecdysone 3β-glucoside,blechnoside A); 2-deoxyecdysone-22-acetate;2-deoxyecdysone-3,22-diacetate; 2-deoxyecdysone-22-β-D-glucopyranoside;2-deoxyecdysone 25-β-D-glucopyranoside; 2-deoxy-21-hydroxyecdysone;3-epi-22-iso-ecdysone; 3-dehydro-2-deoxyecdysone (silenosterone);3-dehydroecdysone; 3-dehydro-2-deoxyecdysone-22-acetate;ecdysone-6-carboxymethyloxime; ecdysone-2,3-acetonide;14-epi-20-hydroxyecdysone-2,3-acetonide;20-hydroxyecdysone-2,3-acetonide; 20-hydroxyecdysone-20,22-acetonide;14-epi-20-hydroxyecdysone-2,3,20,22-diacetonide;paxillosterone-20,22-p-hydroxybenzylidene acetal; poststerone;(20R)-dihydropoststerone; (20S)dihydropoststerone;poststerone-20-dansylhydrazine;(20S)-dihydropoststerone-2,3,20-tribenzoate;(20R)-dihydropoststerone-2,3,20-tribenzoate;(20R)dihydropoststerone-2,3-acetonide;(20S)dihydropoststerone-2,3-acetonide; (5α-H)-dihydrorubrosterone;2,14,22,25-tetradeoxy-5α-ecdysone; 5α-ketodiol, bombycosterol;2α,3α,22S,25-tetrahydroxy-5α-cholestan-6-one;(5α-H)-2-deoxy-21-hydroxyecdysone; castasterone; 24-epi-castasterone;(5α□-H)-2-deoxyintegristerone A; (5α-H)-22-deoxyintegristerone A;(5α-H)-20-hydroxyecdysone; 24,25-didehydrodacryhaninansterone;25,26-didehydrodacryhainansterone; 5-deoxykaladasterone(dacryhainansterone); (14α-H)-14-deoxy-25-hydroxydacryhainansterone;25-hydroxydacryhainansterone; rubrosterone; (5β-H)-dihydrorubrosterone;dihydrorubrosterone-17β-acetate; sidisterone;20-hydroxyecdysone-2,3,22-triacetate;14-deoxy(14β-H)-20-hydroxyecdysone; 14-epi-20-hydroxyecdysone;9α,20-dihydroxyecdysone; malacosterone, 2-deoxypolypodineB-3-β-D-glucopyranoside; ajugalactone; cheilanthone B;2β,3β,6α-trihydroxy-5β-cholestane; 2β,3β,6β-trihydroxy-5β-cholestane;14-dehydroshidasterone; stachysterone B;2β,3β,9α,20R,22R,25-hexahydroxy-5β-cholest-7,14-dien-6-one;kaladasterone; (14β-H)-14-deoxy-25-hydroxydacryhainansterone;4-dehydro-20-hydroxyecdysone; 14-methyl-12-en-shidasterone;14-methyl-12-en-15,20-dihydroxyecdysone; podecdysone B;2β,3β,20R,22R-tetrahydroxy-25-fluoro-5β-cholest-8,14-dien-6-one(25-fluoropodecdysone B); calonysterone;14-deoxy-14,18-cyclo-20-hydroxyecdysone;9α,14α-epoxy-20-hydroxyecdysone; 9βα,14β-epoxy-20-hydroxyecdysone;9α,14α-epoxy-20-hydroxyecdysone 2,3,20,22-diacetonide;28-homobrassinolide; and iso-homobrassinolide.

The disclosure of all patents, patent applications, and publicationscited herein are incorporated by reference in their entireties.

The following examples are illustrative, but not limiting, of themethods of the present invention. Other suitable modifications andadaptations of the variety of conditions and parameters normallyencountered in medical treatment and gene expression systems and whichare obvious to those skilled in the art are within the spirit and scopeof the invention.

Pharmaceutical Compositions

In certain embodiments, polynucleotides and polypeptides of theinvention can be administered as part of a medicament or pharmaceuticalcomposition. Medicaments and pharmaceutical compositions of theinvention comprise one or more pharmaceutically acceptable carriers,diluents, excipients or additives.

The term “excipient” as used herein is typically an inert substanceadded to a composition to facilitate processing, handling,administration, et cetera of a pharmaceutically acceptable composition.Useful excipients include, but are not limited to, adjuvants,anti-adherents, binders, carriers, disintegrants, fillers, flavors,colors, diluents, lubricants, glidants, preservatives, sorbents,solvents, surfactants, and sweeteners.

A few examples of pharmaceutically acceptable carriers, diluents,excipients and additives include, without limitation, water, saline,Ringer's solution, dextrose solution, buffers (such as phosphates (e.g.,calcium phosphates such as tricalcium phosphate or calcium hydrogenphosphate)), citrate, succinate, acetic acid, and other organic acids ortheir salts), antioxidants, proteins and other high molecular weightmolecules (such as serum albumin, gelatin, or immunoglobulins),hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (suchas glycine, glutamic acid, aspartic acid, and arginine), saccharides(for example monosaccharides, disaccharides, lactose, sucrose, mannitol,sorbitol, other carbohydrates and sugar-alcohols, cellulose or itsderivatives, glucose, mannose, and dextrins), chelating agents (such asEDTA); sugar alcohols (such as mannitol or sorbitol), counterions (suchas sodium), surfactants (such as polysorbates, poloxamers, orpolyethylene glycol (PEG)), and binders (such as starch paste (e.g.,maize starch, wheat starch, rice starch, potato starch)), gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone).

Pharmaceutically acceptable carriers, diluents, excipients and additivesmay include: disintegrating agents such as the above-mentioned starchesas well as compounds such as carboxymethyl-starch, cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such assodium alginate; and, flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate, and/or polyethylene glycol. In oneembodiment, dragee cores are provided with suitable coatings which, ifdesired, are resistant to gastric juices. For this purpose, concentratedsaccharide solutions may be used, which may optionally contain gumarabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titaniumdioxide, lacquer solutions and suitable organic solvents or solventmixtures. In order to produce coatings resistant to gastric juices,solutions of suitable cellulose preparations such as acetylcellulosephthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, forexample, for identification or in order to characterize combinations ofactive compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules ornanoparticles which may optionally be mixed with fillers such aslactose, binders such as starches, and/or lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In one embodiment, theis dissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin, optionally with stabilizers.

Fatty oils may comprise mono-, di- or triglycerides. Mono-, di- andtriglycerides include those that are derived from C6, C8, C10, C12, C14,C16, C18, C20 and C22 acids. Exemplary diglycerides include, inparticular, diolein, dipalmitolein, and mixed caprylin-caprindiglycerides. Preferred triglycerides include vegetable oils, fish oils,animal fats, hydrogenated vegetable oils, partially hydrogenatedvegetable oils, synthetic triglycerides, modified triglycerides,fractionated triglycerides, medium and long-chain triglycerides,structured triglycerides, and mixtures thereof. Exemplary triglyceridesinclude: almond oil; babassu oil; borage oil; blackcurrant seed oil;canola oil; castor oil; coconut oil; corn oil; cottonseed oil; eveningprimrose oil; grapeseed oil; groundnut oil; mustard seed oil; olive oil;palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil;sesame oil; shark liver oil; soybean oil; sunflower oil; hydrogenatedcastor oil; hydrogenated coconut oil; hydrogenated palm oil;hydrogenated soybean oil; hydrogenated vegetable oil; hydrogenatedcottonseed and castor oil; partially hydrogenated soybean oil; partiallysoy and cottonseed oil; glyceryl tricaproate; glyceryl tricaprylate;glyceryl tricaprate; glyceryl triundecanoate; glyceryl trilaurate;glyceryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate;glyceryl tricaprylate/caprate; glyceryl tricaprylate/caprate/laurate;glyceryl tricaprylate/caprate/linoleate; and glyceryltricaprylate/caprate/stearate.

In one embodiment, the triglyceride is the medium chain triglycerideavailable under the trade name LABRAFAC CC. Other triglycerides includeneutral oils, e.g., neutral plant oils, in particular fractionatedcoconut oils such as known and commercially available under the tradename MIGLYOL, including the products: MIGLYOL 810; MIGLYOL 812; MIGLYOL818; and CAPTEX 355. Other triglycerides are caprylic-capric acidtriglycerides such as known and commercially available under the tradename MYRITOL, including the product MYRITOL 813. Further triglyceridesof this class are CAPMUL MCT, CAPTEX 200, CAPTEX 300, CAPTEX 800, NEOBEEM5 and MAZOL 1400.

Pharmaceutical compositions comprising triglycerides may furthercomprise lipophilic and/or hydrophilic surfactants which may form clearsolutions upon dissolution with an aqueous solvent. One such surfactantis tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS).Examples of such compositions are described in U.S. Pat. No. 6,267,985.

In another embodiment, the pharmaceutically acceptable carrier comprisesLABRASOL (Gattefosse SA), which is PEG-8 caprylic/capric glycerides. Inanother embodiment, the pharmaceutically acceptable carrier comprisesPL90G, vitamin E TPGS, and Miglyol 812N.

Pharmaceutical compositions can be administered in any suitable manneras determined by those skilled in the art, such as, but withoutlimitation, by oral, rectal, vaginal, topical (including dermal, buccaland sublingual), parenteral, intravenous, intraperitoneal,intramuscular, intratumoral, intraarticular, subcutaneous, intranasal,inhalation, intradermal, intrathecal, epidural, and by naso-gastricroutes.

Methods and compositions for preparation, formulation, and delivery ofpharmaceutically acceptable compositions and medicaments are well-knownand routinely practiced by those skilled in the art. A few examples oftextbooks and manuals providing information and instruction on suchmethods and compositions include: Rowe et al. (Editor), “Handbook ofPharmaceutical Excipients,” Pharmaceutical Press, 6^(th) Ed. (August2009); University of the Sciences in Philadelphia (Editor), “Remington:The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins,21^(st) Ed. (2005); “Physicians' Desk Reference 2011,” PDR Network(2010); “Physicians' Desk Reference 2012,” PDR Network (2011); O'Neil,“The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals,”14^(th) Ed. (2006); Allen et al. (Editor) “Ansel's Pharmaceutical DosageForms and Drug Delivery Systems,” Lippincott Williams & Wilkins; 9^(th)Ed. (2011); and, Ash et al. (Editor), “Handbook of PharmaceuticalAdditives, Third Edition,” Synapse Information Resources, Inc.; 3^(rd)Ed. (2007).

Protocols for general molecular biology methods can be found in: Methodsin Molecular Biology, series editor J M Walker, Humana Press, New York.

Embodiments of the invention comprise any amino acid substituted form ofPE as indicated by, or represented in, Table 13. Embodiments of theinvention further comprise any amino acid substituted form of PE whichmay comprise any combination of amino acid substitutions indicated by,or represented in, Table 13.

Embodiments of the invention also comprise variants, derivatives, orbiologically active fragments of any amino acid substituted form of PEas indicated by, or represented in, Table 13, wherein said variant,derivative, or biologically active fragment of PE is at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 97% identical, at least 98% identical, at least 99%identical, or is at least 100% identical to an amino acid substitutedform of PE, or a fragment thereof, as indicated by, or represented in,Table 13. For example, embodiments of the invention comprise variants,derivatives, or biologically active fragments of any amino acidsubstituted form of PE as indicated by, or represented in, Table 13,wherein said variant, derivative, or biologically active fragment of PEis at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 97% identical, at least 98%identical, at least 99% identical, or is at least 100% identical to PEconstructs, or fragments thereof, as represented by pIEX02-003 throughpIEX02-248 in Table 13 (such as, for example, as shown in SEQ ID NO:177(pIEX02-228), SEQ ID NO:178 (pIEX02-244), and SEQ ID NO: 179(pIEX02-246)).

Embodiments of the invention include methods of making, methods ofusing, methods of treatment using, medicaments comprising,pharmaceutically acceptable compositions comprising, therapeuticallyuseful compositions comprising, and kits comprising any of the aminoacid substituted forms of PE referenced, or otherwise described orprovided for, herein.

Embodiments of the invention also include (where “E” indicates“embodiment”):

-   -   E1. An isolated polypeptide having Pseudomonas exotoxin A        biological activity, wherein said polypeptide comprises an        epitope selected from the group consisting of:

a) ISFSTRGTQ; (SEQ ID NO: 5) b) GTQNWTVER; (SEQ ID NO: 6) c) IVFGGVRAR;(SEQ ID NO: 7) d) ARSQDLDAI; (SEQ ID NO: 8) e) LRVYVPRSS; (SEQ ID NO: 9)f) IPDKEQAIS; (SEQ ID NO: 10) g) ISFSTRGTQNWTVER; (SEQ ID NO: 131) andh) IVFGGVRARSQDLDAI (SEQ ID NO: 132)wherein one or more amino acid residues in any one or more of saidepitopes in a) through h) are substituted with a different amino acidresidue.

-   -   E2. An isolated polypeptide having Pseudomonas exotoxin A        biological activity, wherein said polypeptide comprises an        epitope selected from the group consisting of:        -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino acid residues at            one or more of positions 1, 6 and 9 are substituted with a            different amino acid residue;        -   b) GTQNWTVER (SEQ ID NO:6), wherein amino acid residues at            one or more of positions 3, 4 and 6 are substituted with a            different amino acid residue;        -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino acid residues at            one or more of positions 1 and 6 are substituted with a            different amino acid residue;        -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino acid residues at            one or more of positions 4 and 7 are substituted with a            different amino acid residue;        -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino acid residues at            one or more of positions 1, 2 and 9 are substituted with a            different amino acid residue;        -   f) IPDKEQAIS (SEQ ID NO: 10), wherein amino acid residues at            one or more of positions 1, 4, 6 and 7 are substituted with            a different amino acid residue;        -   g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein amino acid            residues at one or more of positions 1, 6, 9, 10 and 12 are            substituted with a different amino acid residue; and        -   h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein amino acid            residues at one or more of positions 1, 6, 11, and 14 are            substituted with a different amino acid residue.    -   E3. The isolated polypeptide of embodiment E1 or E2, wherein        said different amino acid residue is a conservative amino acid        substitution.    -   E4. The isolated polypeptide of embodiment E3, wherein said        conservative amino acid substitution is one or more        substitutions selected from the group consisting of:        -   a) A is substituted with any one of G, I, L, S, T or V;        -   b) D is substituted with E;        -   c) I is substituted with any one of L, M or V;        -   d) K is substituted with any one of H or R;        -   e) L is substituted with any one of A, G, I, M or V;        -   f) N is substituted with any one of S, T or Q;        -   g) Q is substituted with any one of S, T or N,        -   h) R is substituted with any one of K or H;        -   i) S is substituted with any one of A, G, N, T or Q;        -   j) T is substituted with any one of A, G, N, Q or S; and        -   k) V is substituted with any one of A, G, I, L or M.    -   E5. An isolated polypeptide having Pseudomonas exotoxin A        biological activity, wherein said polypeptide comprises an        epitope selected from the group consisting of:        -   a) ISFSTRGTQ (SEQ ID NO:5), wherein amino the acid residue            at position 1 (I) is substituted with A, N, T, Q or H, or            wherein the amino acid residue at position 6 (R) is            substituted with Q, or wherein the amino acid residue at            position 9 (Q) is substituted with N or T, or wherein the            amino acid sequence ISFSTRGTQ (SEQ ID NO:5) comprises two or            more of said substitutions in any combination;        -   b) GTQNWTVER (SEQ ID NO:6), wherein the amino acid residue            at position 3 (Q) is substituted with N or T, wherein amino            the acid residue at position 4 (N) is substituted with K or            R, or wherein the amino acid residue at position 6 (T) is            substituted with K or R, or wherein the amino acid sequence            GTQNWTVER (SEQ ID NO:6) comprises two or more of said            substitutions in any combination;        -   c) IVFGGVRAR (SEQ ID NO:7), wherein amino the acid residue            at position 1 (1) is substituted with A or N, or wherein the            amino acid residue at position 6 (V) is substituted with D,            M, or N, or wherein the amino acid sequence IVFGGVRAR (SEQ            ID NO:7) comprises substitutions at both positions in any            combination of amino acid residues A or N at position 1 (I)            and D, M, or N at position 6 (V);        -   d) ARSQDLDAI (SEQ ID NO:8), wherein amino the acid residue            at position 4 (Q) is substituted with K or R, or wherein the            amino acid residue at position 7 (D) is substituted with K            or R, or wherein the amino acid sequence ARSQDLDAI (SEQ ID            NO:8) comprises substitutions with K or R in any combination            at both positions 4 (Q) and 7 (D);        -   e) LRVYVPRSS (SEQ ID NO:9), wherein amino the acid residue            at position 1 (L) is substituted with A, or wherein the            amino acid residue at position 2 (R) is substituted with D,            S or A, or wherein the amino acid residue at position 9 (S)            is substituted with D, E, N, K, P or T, or wherein the amino            acid sequence LRVYVPRSS (SEQ ID NO:9) comprises two or more            of said substitutions in any combination;        -   f) IPDKEQAIS (SEQ ID NO:10), wherein amino acid residues at            one or more of positions 1, 4, 6 and 7 are substituted with            a different amino acid residue, wherein amino the acid            residue at position 1 (I) is substituted with A, N, T, Q or            H, or wherein the amino acid residue at position 4 (K) is            substituted with T, or wherein the amino acid residue at            position 6 (Q) is substituted with D, or wherein the amino            acid residue at position 7 (A) is substituted with D, or            wherein the amino acid sequence IPDKEQAIS (SEQ ID NO: 10)            comprises two or more of said substitutions in any            combination;        -   g) ISFSTRGTQNWTVER (SEQ ID NO:131), wherein amino acid            residues at one or more of positions 1, 6, 9, 10 and 12 are            substituted with a different amino acid residues wherein            amino the acid residue at position 1 (I) is substituted with            A, N, T, Q or H, or wherein the amino acid residue at            position 6 (R) is substituted with Q, or wherein the amino            acid residue at position 9 (Q) is substituted with N or T,            or wherein amino the acid residue at position 10 (N) is            substituted with K or R, or wherein the amino acid residue            at position 12 (T) is substituted with K or R, or wherein            the amino acid sequence ISFSTRGTQNWTVER (SEQ ID NO: 131)            comprises two or more of said substitutions in any            combination; and        -   h) IVFGGVRARSQDLDAI (SEQ ID NO:132), wherein amino the acid            residue at position 1 (I) is substituted with A or N, or            wherein the amino acid residue at position 6 (V) is            substituted with D, M, or N, wherein amino the acid residue            at position 11 (Q) is substituted with K or R, or wherein            the amino acid residue at position 14 (D) is substituted            with K or R, or wherein the amino acid sequence            IVFGGVRARSQDLDAI (SEQ ID NO:132) comprises two or more of            said substitutions in any combination.    -   E6. An isolated polypeptide having Pseudomonas exotoxin A        biological activity, wherein said polypeptide comprises an        epitope selected from the group consisting of:        -   a) I at position 141 is a different amino acid;        -   b) R at position 146 is a different amino acid;        -   c) Q at position 149 is a different amino acid;        -   d) N at position 150 is a different amino acid;        -   e) T at position 152 is a different amino acid;        -   f) I at position 184 is a different amino acid;        -   g) V at position 189 is a different amino acid;        -   h) Q at position 194 is a different amino acid;        -   i) D at position 197 is a different amino acid;        -   j) L at position 233 is a different amino acid;        -   k) R at position 234 is a different amino acid;        -   l) S at position 241 is a different amino acid;        -   m) I at position 321 is a different amino acid;        -   n) K at position 324 is a different amino acid;        -   o) Q at position 326 is a different amino acid;        -   p) A at position 327 is a different amino acid;        -   q) any combination of one or more of a) through p),    -   wherein the amino acid numbering corresponds to SEQ ID NO: 1.    -   E7. An isolated polypeptide comprising an amino acid sequence        identical to SEQ ID NO:1, except for one or more amino acid        substitutions selected from the group consisting of:        -   a) I at position 141 is substituted with a conservative            amino acid substitution;        -   b) R at position 146 is substituted with a conservative            amino acid substitution;        -   c) Q at position 149 is substituted with a conservative            amino acid substitution;        -   d) N at position 150 is substituted with a conservative            amino acid substitution;        -   e) T at position 152 is substituted with a conservative            amino acid substitution;        -   f) I at position 184 is substituted with a conservative            amino acid substitution;        -   g) V at position 189 is substituted with a conservative            amino acid substitution;        -   h) Q at position 194 is substituted with a conservative            amino acid substitution d;        -   i) D at position 197 is substituted with a conservative            amino acid substitution;        -   j) L at position 233 is substituted with a conservative            amino acid substitution;        -   k) R at position 234 is substituted with a conservative            amino acid substitution;        -   l) S at position 241 is substituted with a conservative            amino acid substitution;        -   m) I at position 321 is substituted with a conservative            amino acid substitution;        -   n) K at position 324 is substituted with a conservative            amino acid substitution;        -   o) Q at position 326 is substituted with a conservative            amino acid substitution;        -   p) A at position 327 is substituted with a conservative            amino acid substitution;        -   q) any combination of one or more of a) through p),    -   wherein the amino acid numbering corresponds to SEQ ID NO: 1.    -   E8. The isolated polypeptide of embodiment E7, wherein said        conservative amino acid substitution is one or more        substitutions selected from the group consisting of:        -   a) A is substituted with any one of G, I, L, S, T or V;        -   b) D is substituted with E;        -   c) I is substituted with any one of L, M or V;        -   d) K is substituted with any one of H or R;        -   e) L is substituted with any one of A, G, I, M or V;        -   f) N is substituted with any one of S, T or Q;        -   g) Q is substituted with any one of S, T or N;        -   h) R is substituted with any one of K or H;        -   i) S is substituted with any one of A, G, N, T or Q;        -   j) T is substituted with any one of A, G, N, Q or S; and        -   k) V is substituted with any one of A, G, I, L or M.    -   E9. An isolated polypeptide comprising an amino acid sequence        identical to SEQ ID NO:1, except for one or more amino acid        substitutions selected from the group consisting of:

a) I at position 141 is A; b) I at position 141 is N; c) I at position141 is T; d) I at position 141 is Q; e) I at position 141 is H; f) R atposition 146 is Q; g) Q at position 149 is N; h) Q at position 149 is T;i) N at position 150 is R; j) N at position 150 is K; k) T at position152 is R; l) T at position 152 is K; m) I at position 184 is A; n) I atposition 184 is N; o) V at position 189 is D; p) V at position 189 is M;q) V at position 189 is N; r) Q at position 194 is R; s) Q at position194 is K; t) D at position 197 is R; u) D at position 197 is K; v) L atposition 233 is A; w) R at position 234 is D; x) R at position 234 is S;y) Rat position 234 is A; z) S at position 241 is D; ab) S at position241 is E; ac) S at position 241 is N; ad) S at position 241 is K; ae) Sat position 241 is P; af) S at position 241 is T; ag) I at position 321is A; ah) I at position 321 is N; ai) I at position 321 is T; ak) I atposition 321 is Q; al) I at position 321 is H; am) K at position 324 isT; an) Q at position 326 is D; ao) A at position 327 is D; ap) anycombination of one or more of a) through ao),wherein the amino acid numbering corresponds to SEQ ID NO: 1.

-   -   E10. The polypeptide in any one of embodiments E1 to E9,        comprising a number of amino acid substitutions selected from        the group consisting of:

a)  1 amino acid substitution; b)  2 amino acid substitutions; c)  3amino acid substitutions; d)  4 amino acid substitutions; e)  5 aminoacid substitutions; f)  6 amino acid substitutions; g)  7 amino acidsubstitutions; h)  8 amino acid substitutions; i)  9 amino acidsubstitutions; j) 10 amino acid substitutions; k) 11 amino acidsubstitutions; l) 12 amino acid substitutions; m) 13 amino acidsubstitutions; n) 14 amino acid substitutions; o) 15 amino acidsubstitutions; and p) 16 amino acid substitutions.

-   -   E11. The polypeptide of embodiment E9, comprising amino acid        substitutions present at each of amino acid positions 141, 146,        149, 150, 152, 184, 189, 194, 197, 233, 234, 241, 321, 324, 326        and 327.    -   E12. The polypeptide of any one of embodiments E1 to E11,        wherein said polypeptide comprises the amino acid sequence in        SEQ ID NO:1, except for amino acid substitutions indicated in        embodiments E1 to E11.    -   E13. The polypeptide in any one of embodiments E1 to E11,        wherein said polypeptide is a variant or fragment of a        Pseudomonas exotoxin A polypeptide.    -   E14. The polypeptide of embodiment E13, wherein said variant or        fragment comprises a number of epitopes selected from the group        consisting of:        -   a) at least one epitope;        -   b) at least two epitopes;        -   c) at least three epitopes;        -   d) at least four epitopes;        -   e) at least five epitopes; and        -   f) at least six epitopes.    -   E15. The polypeptide of embodiment E14, wherein said polypeptide        comprises a contiguous number of amino acids selected from the        group consisting of:        -   a) at least 20 contiguous amino acids;        -   b) at least 30 contiguous amino acids;        -   c) at least 40 contiguous amino acids;        -   d) at least 50 contiguous amino acids;        -   e) at least 60 contiguous amino acids;        -   f) at least 70 contiguous amino acids.        -   g) at least 80 contiguous amino acids;        -   h) at least 90 contiguous amino acids;        -   i) at least 100 contiguous amino acids;        -   j) at least 125 contiguous amino acids;        -   k) at least 150 contiguous amino acids;        -   l) at least 175 contiguous amino acids.        -   m) at least 200 contiguous amino acids;        -   n) at least 225 contiguous amino acids;        -   o) at least 250 contiguous amino acids;        -   p) at least 275 contiguous amino acids;        -   q) at least 300 contiguous amino acids;        -   r) at least 325 contiguous amino acids; and        -   s) at least 350 contiguous amino acids.    -   E16. An isolated polypeptide comprising a Pseudomonas exotoxin A        (PE-A) cytotoxic domain (Domain III), wherein the cytotoxic        domain comprises one or more amino acid substitutions which        prevent or reduce host immunogenic responses compared to the        same polypeptide without said one or more amino acid        substitutions.    -   E17. The polypeptide of embodiment E16, wherein said one or more        amino acid substitutions are introduced into a cytotoxic domain        sequence selected from the group consisting of:        -   (a) amino acid residues Phe-134 to Lys-347 of SEQ ID NO: 1;        -   (b) amino acid residues Phe-134 to Lys-347 of SEQ ID NO:4;        -   (c) amino acid residues Phe-400 to Lys-613 of SEQ ID NO:133;            and        -   (d) amino acid residues Phe-400 to Lys-613 of SEQ ID NO:134.    -   E18. The polypeptide of embodiment E17, wherein the last five        amino acids in said cytotoxic domain are replaced with an amino        acid sequence selected from the group consisting of:

(i) Arg-Glu-Asp-Leu; (SEQ ID NO: 136) and (ii) Lys-Asp-Glu-Leu.(SEQ ID NO: 137)

-   -   E19. The polypeptide in any one of embodiments E16 to E18,        wherein said polypeptide further comprises one or more PE-A        domains selected from the group consisting of:        -   (a) a cytosolic translocation domain (Domain II);        -   (b) a carboxy-terminal portion of Domain IB;        -   (c) an amino-terminal portion of Domain IB; and        -   (d) a complete Domain IB.    -   wherein one or more of said domains has been modified with amino        acid substitutions, as described herein, to reduce or eliminate        immunogenicity.    -   E20. The polypeptide of embodiment E19, wherein said cytosolic        translocation domain (Domain II) comprises an amino acid        sequence selected from the group consisting of:        -   (a) amino acids Gly-3 to Ser-114 of SEQ ID NO:1; and        -   (b) amino acids Gly-3 to Asn-114 of SEQ ID NO:4.    -   E21. The polypeptide of embodiment E19, wherein said        carboxy-terminal portion of Domain IB comprises the amino acid        sequence of Gly-115 to Glu-133 of SEQ ID NO: 1 or wherein said        amino-terminal portion of Domain IB comprises the amino acid        sequence of Ala-365 to Ala-380 of SEQ ID NO:133.    -   E22. The polypeptide of embodiment E19, wherein said complete        Domain IB comprises the amino acid sequence of Ala-365 to        Glu-399 of SEQ ID NO:133.    -   E23. The polypeptide in any one of embodiments E16 to E22,        wherein said polypeptide is a variant or fragment of a        Pseudomonas exotoxin A polypeptide.    -   E24. The polypeptide of any one of embodiments E1 to E23,        wherein said polypeptide has one or more biological activities        selected from the group consisting of:        -   a) eukaryotic cell killing activity (cell cytotoxicity);        -   b) inhibits translation elongation factor EF-2 biological            activity;        -   c) induces or catalyzes ADP-ribosylation of EF-2; and        -   d) inhibits protein synthesis.    -   E25. The polypeptide of any one of embodiments E1 to E24,        wherein said one or more amino acid substitutions prevent or        reduce host immunogenic responses compared to the same        polypeptide without the corresponding said one or more amino        acid substitutions.    -   E26. The polypeptide of any one of embodiments E1 to E24,        wherein said one or more amino acid substitutions prevent or        reduce host immunogenic responses compared to a polypeptide        comprising an amino acid sequence selected from the group        consisting of:        -   (a) SEQ ID NO:1;        -   (b) SEQ ID NO:4;        -   (c) SEQ ID NO:133; and        -   (d) SEQ ID NO:134.    -   E27. The polypeptide of any one of embodiments E1 to E26,        wherein said polypeptide is a fusion protein.    -   E28. The fusion protein of embodiment E27, wherein the        amino-terminal end of said polypeptide in any one of embodiments        E1 to E26 is fused to the carboxyl-terminal end of a different        polypeptide.    -   E29. The fusion protein of embodiment E27, wherein the        carboxyl-terminal end of said polypeptide in any one of        embodiments E1 to E26 is fused to the amino-terminal end of a        different polypeptide.    -   E30. The fusion protein in embodiment E28 or E29, wherein said        different polypeptide comprises an antigen binding moiety.    -   E31. The fusion protein of embodiment E30, wherein said antigen        binding moiety is an antibody or fragment thereof.    -   E32. The fusion protein of embodiment E31, wherein said        antibody, or fragment thereof, is an antibody selected from the        list in Table 1, or is a fragment thereof.    -   E33. The fusion protein of embodiment E31, wherein said        antibody, or fragment thereof, specifically binds to a        cancer-specific or tumor-specific antigen.    -   E34. The fusion protein of embodiment E33, wherein said        cancer-specific or tumor-specific antigen is a breast cancer        antigen.    -   E35. The fusion protein of embodiment E34, wherein said breast        cancer antigen is HER2.    -   E36. The fusion protein of embodiment E31, wherein said        antibody, or fragment thereof is selected from the group        consisting of:        -   a) ERTUMAXOMAB (Rexomun);        -   b) PERTUZUMAB (Omnitarg); and        -   c) TRASTUZUMAB (Herceptin).    -   E37. The fusion protein of any one of embodiments E27 to E29,        wherein said different polypeptide comprises a polypeptide        selected from the group consisting of:        -   a) Mesothelin;        -   b) CD24;        -   c) CD22;        -   d) CD25;        -   e) CD174;        -   f) TPBG;        -   g) CD56; and        -   h) C-type lectin-like molecule-1.    -   E38. An isolated polynucleotide encoding the polypeptide or        fusion protein in any one of embodiments E1 to E37.    -   E39. An expression vector comprising the polynucleotide of        embodiment E38.    -   E40. A host cell comprising the expression vector of embodiment        E39.    -   E41. A method of producing the polypeptide or fusion protein in        any one of embodiments E1 to E37, wherein said method comprises:        -   a) obtaining a host cell comprising a polynucleotide            encoding said polypeptide or fusion protein;        -   b) exposing said host cell to conditions wherein said            polypeptide or fusion protein is produced.    -   E42. A method of producing the polypeptide or fusion protein in        any one of embodiments E1 to E37, wherein said method comprises        use of an expression system comprising:        -   (A) a first polynucleotide encoding a first hybrid            polypeptide comprising:            -   (i) a first ligand binding domain; and            -   (ii) a DNA-binding domain;        -   (B) a second polynucleotide encoding a second hybrid            polypeptide comprising:            -   (i) a second ligand binding domain; and            -   (ii) a transactivation domain;        -   (C) a third polynucleotide encoding the polypeptide or            fusion protein in any one of embodiments E1 to E37, wherein            said third polynucleotide is operably associated with a            response element capable of being bound by the DNA-binding            domain of said first hybrid polypeptide;    -   wherein the first ligand binding domain and the second ligand        binding domain are capable of ligand-induced dimerization,    -   wherein expression of the polypeptide or fusion protein in any        one of embodiments E1 to E37 is modulated by a ligand which        induces dimerization of said first and said second ligand        binding domains,    -   wherein the polypeptide or fusion protein in any one of        embodiments E1 to E37 is produced by allowing said ligand to        contact said first and said second ligand binding domains.    -   E43. A single expression vector or two or more expression        vectors comprising the first, second, and third polynucleotides        of embodiment E42.    -   E44. The expression vector or expression vectors of embodiment        E43, wherein one or more of the vectors is a viral expression        vector.    -   E45. A host cell comprising the expression vector or expression        vectors of embodiments E43 or E44.    -   E46. A method of treating a disease or disorder comprising        administering to a subject in need thereof the polypeptide or        fusion protein in any one of embodiments E1 to E37, the        polynucleotide of embodiment E38, the vector of embodiment E39,        the host cell of embodiment E40, or a polypeptide or fusion        protein produced by the method of embodiment E41.    -   E47. A method of treating a disease or disorder comprising        delivering to a subject in need thereof a polypeptide or fusion        protein produced by the method of embodiment E42, wherein said        method comprises administration of the ligand to said subject.    -   E48. The method of embodiment E47, wherein the polypeptide or        fusion protein is delivered to the subject by first        administering the first, second, and third polynucleotides.    -   E49. The method of embodiment E47, wherein the polypeptide or        fusion protein is delivered to the subject by first        administering the expression vector or expression vectors of        embodiments E43 or E44.    -   E50. The method of embodiment E47, wherein said polypeptide or        fusion protein is delivered to the subject by first        administering the host cell of embodiment E45.    -   E51. A pharmaceutical composition comprising the polypeptide or        fusion protein in any one of embodiments E1 to E37, comprising        the polynucleotide of embodiment E38, comprising the expression        vector or expression vectors in any one of embodiments E39, E43        or E44, or comprising the host cell of embodiments E40 or E45,        and a pharmaceutically acceptable carrier, diluent or excipient.    -   E52. A medicament comprising the polypeptide or fusion protein        in any one of embodiments E1 to E37, comprising the        polynucleotide of embodiment E38, comprising the expression        vector or expression vectors in any one of embodiments E39, E43        or E44, or comprising the host cell of embodiments E40 or E45.    -   E53. Use of the medicament of embodiment E52, wherein said use        is for the treatment of a disease or disorder.    -   E54. Use of the medicament according to embodiment E53, wherein        the disease or disorder is cancer.    -   E55. A polypeptide having at least one Pseudomonas exotoxin A        (PE-A) biological activity, wherein said polypeptide comprises        one or more amino acid substitutions compared to a wild-type        PE-A polypeptide, wherein said one or more amino acid        substitutions is a substitution of a different amino acid at one        or more positions corresponding to amino acid residues in the        polypeptide of SEQ ID NO: 1, wherein said substitution positions        are selected from the group consisting of:        -   a) isoleucine (I) at position 141;        -   b) arginine (R) at position 146;        -   c) glycine (G) at position 147;        -   d) glutamine (Q) at position 149;        -   e) asparagine (N) at position 150;        -   f) threonine (T) at position 152;        -   g) valine (V) at position 189;        -   h) arginine (R) at position 192;        -   i) glutamine (Q) at position 194;        -   j) aspartic acid (D) at position 197;        -   k) serine (S) at position 241;        -   l) isoleucine (I) at position 321; and        -   m) glutamine (Q) at position 326.    -   E56. The polypeptide of embodiment E55, wherein said one or more        amino acid substitutions is a conservative amino acid        substitution.    -   E57. The polypeptide of embodiment E55, wherein said one or more        amino acid substitutions is selected from the group consisting        of:        -   a) isoleucine (I) at position 141 is substituted with            alanine (A), threonine (T), or histidine (H);        -   b) arginine (R) at position 146 is substituted with            glutamine (Q) or alanine (A);        -   c) glycine (G) at position 147 is substituted with serine            (S);        -   d) glutamine (Q) at position 149 is substituted with            threonine (T);        -   e) asparagine (N) at position 150 is substituted with            alanine (A);        -   f) threonine (T) at position 152 is substituted with            alanine (A) or arginine (R);        -   g) valine (V) at 189 is substituted with alanine (A);        -   h) arginine (R) at position 192 is substituted with            alanine (A) or glutamine (Q);        -   i) glutamine (Q) at position 194 is substituted with            arginine (R);        -   j) aspartic acid (D) at position 197 is substituted with            lysine (K);        -   k) serine (S) at position 241 is substituted with threonine            (T), asparagine (N), lysine (K), or proline (P);        -   l) isoleucine (I) at position 321 is substituted with            alanine (A), asparagine (N), histidine (H), threonine (T),            or glutamine (Q); and        -   m) glutamine (Q) at position 326 is substituted with            glutamic acid (E).    -   E58. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution for isoleucine (1) at position 141, a        substitution for threonine (T) at position 152, a substitution        for arginine (R) at position 192, a substitution for aspartic        acid (D) at position 197, a substitution for serine (S) at        position 241, and a substitution for glutamine (Q) at position        326.    -   E59. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution of threonine (T) or alanine (A) for        isoleucine (I) at position 141, a substitution alanine (A) or        arginine (R) for threonine (T) at position 152, a substitution        of alanine (A) for arginine (R) at position 192, a substitution        of lysine (K) for aspartic acid (D) at position 197, a        substitution of threonine (T) for serine (S) at position 241,        and a substitution of glutamic acid (E) for glutamine (Q) at        position 326.    -   E60. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution of threonine (T) for isoleucine (I) at        position 141, a substitution alanine (A) for threonine (T) at        position 152, a substitution of alanine (A) for arginine (R) at        position 192, a substitution of lysine (K) for aspartic acid (D)        at position 197, a substitution of threonine (T) for serine (S)        at position 241, and a substitution of glutamic acid (E) for        glutamine (Q) at position 326.    -   E61. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution of alanine (A) for isoleucine (I) at        position 141, a substitution alanine (A) for threonine (T) at        position 152, a substitution of alanine (A) for arginine (R) at        position 192, a substitution of lysine (K) for aspartic acid (D)        at position 197, a substitution of threonine (T) for serine (S)        at position 241, and a substitution of glutamic acid (E) for        glutamine (Q) at position 326.    -   E62. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution for isoleucine (I) at position 141, a        substitution for threonine (T) at position 152, a substitution        for aspartic acid (D) at position 197, a substitution for        serine (S) at position 241, and a substitution for glutamine (Q)        at position 326.    -   E63. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution for isoleucine (I) at position 141, a        substitution for threonine (T) at position 152, a substitution        for arginine (R) at position 192, a substitution for aspartic        acid (D) at position 197, and a substitution for serine (S) at        position 241.    -   E64. The isolated polypeptide of embodiment E55, wherein said        polypeptide comprises a substitution of alanine (A) or        threonine (T) for isoleucine (I) at position 141, a substitution        of arginine (R) or alanine (A) for threonine (T) at position        152, a substitution of lysine (K) for aspartic acid (D) at        position 197, a substitution of threonine (T) for serine (S) at        position 241, and a substitution of glutamic acid (E) for        glutamine (Q) at position 326.    -   E65. The isolated polypeptide of embodiment E55, wherein said        polypeptide comprises a substitution of alanine (A) for        isoleucine (I) at position 141, a substitution of arginine (R)        for threonine (T) at position 152, a substitution of lysine (K)        for aspartic acid (D) at position 197, a substitution of        threonine (T) for serine (S) at position 241, and a substitution        of glutamic acid (E) for glutamine (Q) at position 326.    -   E66. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution of alanine (A) for isoleucine (I) at        position 141, a substitution of alanine (A) for threonine (T) at        position 152, a substitution of lysine (K) for aspartic acid (D)        at position 197, a substitution of threonine (T) for serine (S)        at position 241, and a substitution of glutamic acid (E) for        glutamine (Q) at position 326.    -   E67. The polypeptide of embodiment E55, wherein said polypeptide        comprises a substitution of threonine (T) for isoleucine (I) at        position 141, a substitution of alanine (A) for threonine (T) at        position 152, a substitution of lysine (K) for aspartic acid (D)        at position 197, a substitution of threonine (T) for serine (S)        at position 241, and a substitution of glutamic acid (E) for        glutamine (Q) at position 326.    -   E68. The polypeptide in any one of embodiments E55 to E67,        wherein the at least one Pseudomonas exotoxin A (PE-A)        biological activity comprises the ability to inhibit in vitro        transcription/translation compared to a corresponding wild-type        or non-substituted PE-A polypeptide, wherein said ability to        inhibit in vitro transcription/translation is in an amount        selected from the group consisting of:        -   (a) at least 5% inhibition;        -   (b) at least 10% inhibition;        -   (c) at least 15% inhibition;        -   (d) at least 20% inhibition;        -   (e) at least 25% inhibition;        -   (f) at least 30% inhibition;        -   (g) at least 40% inhibition;        -   (h) at least 50% inhibition;        -   (i) at least 60% inhibition;        -   (j) at least 70% inhibition;        -   (k) at least 80% inhibition;        -   (l) at least 90% inhibition;        -   (m) at least 100% inhibition;        -   (n) about 100% inhibition; and        -   (o) 1000/% inhibition.    -   E69. The polypeptide in any one of embodiments E55 to E68,        comprising a number of amino acid substitutions selected from        the group consisting of:        -   a) 1 amino acid substitution;        -   b) 2 amino acid substitutions;        -   c) 3 amino acid substitutions;        -   d) 4 amino acid substitutions;        -   e) 5 amino acid substitutions; and        -   f) 6 amino acid substitutions.    -   E70. The polypeptide in any one of embodiments E55 to E69,        wherein said polypeptide comprises one or more amino acid        substitutions which prevent or reduce host immunogenic responses        compared to the same polypeptide without said one or more amino        acid substitutions.    -   E71. The polypeptide of embodiment E70, wherein host immunogenic        responses are prevented or reduced in a human host.    -   E72. The polypeptide in any one of embodiments E55 to E71,        wherein the last five or six amino acids in said polypeptide        comprise one or more amino acid sequences selected from the        group consisting of:        -   (i) Arg-Glu-Asp-Leu-Lys;        -   (ii) Arg-Glu-Asp-Leu;        -   (iii) Lys-Asp-Glu-Leu;        -   (iv) Glu-Asp-Leu-Lys; and        -   (v) a dimer, trimer, pentamer, hexamer, septamer, or octamer            of (i), (ii), or (iii), or any combination thereof.    -   E73. The polypeptide of any one of embodiments E55 to E72,        wherein said polypeptide has one or more biological activities        selected from the group consisting of:        -   a) eukaryotic cell killing activity (cell cytotoxicity);        -   b) inhibits translation elongation factor EF-2 biological            activity;        -   c) induces or catalyzes ADP-ribosylation of EF-2; and        -   d) inhibits protein synthesis.    -   E74. The polypeptide of any one of embodiments E55 to E72,        wherein said one or more amino acid substitutions prevent or        reduce host immunogenic responses compared to the same        polypeptide without the corresponding said one or more amino        acid substitutions.    -   E75. A polypeptide comprising a biologically active fragment of        the polypeptide in any one of embodiments E55 to E74.    -   E76. A polypeptide comprising a variant or derivative of the        polypeptide in any one of embodiments E55 to E75, wherein said        variant or derivative shares amino acid sequence identity with        the polypeptide in any one of embodiments E55 to E75, wherein        said shared amino acid sequence identity is selected from the        group consisting of:        -   a) at least 80% identity;        -   b) at least 85% identity;        -   c) at least 90% identity;        -   d) at least 95% identity;        -   e) at least 97% identity;        -   f) at least 98% identity; and        -   g) at least 99% identity.    -   E77. The polypeptide of any one of embodiments E55 to E76,        wherein said one or more amino acid substitutions prevent or        reduce host immunogenic responses compared to a polypeptide        comprising an amino acid sequence selected from the group        consisting of:        -   (a) SEQ ID NO:1;        -   (b) SEQ ID NO:4;        -   (c) SEQ ID NO:133; and        -   (d) SEQ ID NO:134.    -   E78. The polypeptide of any one of embodiments E55 to E77,        wherein said polypeptide is a fusion protein.    -   E79. The fusion protein of embodiment E78, wherein the        amino-terminal end of said polypeptide in any one of embodiments        E55 to E78 is fused to the carboxyl-terminal end of a different        polypeptide.    -   E80. The fusion protein of embodiment E78, wherein the        carboxyl-terminal end of said polypeptide in any one of        embodiments E55 to E78 is fused to the amino-terminal end of a        different polypeptide.    -   E81. The fusion protein in embodiment E79 or E80, wherein said        different polypeptide comprises an antigen binding moiety.    -   E82. The fusion protein of embodiment E81, wherein said antigen        binding moiety is an antibody or fragment thereof.    -   E83. The fusion protein of any one of embodiments E78 to E82,        wherein said antibody, or fragment thereof, is an antibody        selected from the list in Table 1, or is a fragment thereof.    -   E84. The fusion protein of embodiment E82, wherein said        antibody, or fragment thereof, specifically binds to a        cancer-specific or tumor-specific antigen.    -   E85. The fusion protein of embodiment E84, wherein said        cancer-specific or tumor-specific antigen is a breast cancer        antigen.    -   E86. The fusion protein of embodiment E85, wherein said breast        cancer antigen is HER2.    -   E87. The fusion protein of embodiment E82, wherein said        antibody, or fragment thereof is selected from the group        consisting of:        -   a) ERTUMAXOMAB (Rexomun);        -   b) PERTUZUMAB (Omnitarg); and        -   c) TRASTUZUMAB (Herceptin).    -   E88. The fusion protein of any one of embodiments E78 to E80,        wherein said different polypeptide comprises a polypeptide        selected from the group consisting of:        -   a) Mesothelin;        -   b) CD24;        -   c) CD22;        -   d) CD25;        -   e) CD174;        -   f) TPBG;        -   g) CD56; and        -   h) C-type lectin-like molecule-1.    -   E89. A polynucleotide encoding the polypeptide or fusion protein        in any one of embodiments E55 to E88.    -   E90. An expression vector comprising the polynucleotide of        embodiment E89.    -   E91. A host cell comprising the expression vector of embodiment        E90.    -   E92. A method of producing the polypeptide or fusion protein in        any one of embodiments E55 to E88, wherein said method        comprises:        -   a) obtaining a host cell comprising a polynucleotide            encoding said polypeptide or fusion protein;        -   b) exposing said host cell to conditions wherein said            polypeptide or fusion protein is produced.    -   E93. A method of producing the polypeptide or fusion protein in        any one of embodiments E55 to E88, wherein said method comprises        use of an expression system comprising:        -   (A) a first polynucleotide encoding a first hybrid            polypeptide comprising:            -   (i) a first ligand binding domain; and            -   (ii) a DNA-binding domain;        -   (B) a second polynucleotide encoding a second hybrid            polypeptide comprising:            -   (i) a second ligand binding domain; and            -   (ii) a transactivation domain;        -   (C) a third polynucleotide encoding the polypeptide or            fusion protein in any one of embodiments E55 to E88, wherein            said third polynucleotide is operably associated with a            response element capable of being bound by the DNA-binding            domain of said first hybrid polypeptide;    -   wherein the first ligand binding domain and the second ligand        binding domain are capable of ligand-induced dimerization,    -   wherein expression of the polypeptide or fusion protein in any        one of embodiments E55 to E88 is modulated by a ligand which        induces dimerization of said first and said second ligand        binding domains,    -   wherein the polypeptide or fusion protein in any one of        embodiments E55 to E88 is produced by allowing said ligand to        contact said first and said second ligand binding domains.    -   E94. A single expression vector or two or more expression        vectors comprising the first, second, and third polynucleotides        of embodiment E93.    -   E95. The expression vector or expression vectors of embodiment        E94, wherein one or more of the vectors is a viral expression        vector.    -   E96. A host cell comprising the expression vector or expression        vectors of embodiments E94 or E95.    -   E97. A method of treating a disease or disorder comprising        administering to a subject in need thereof the polypeptide or        fusion protein in any one of embodiments E55 to E88, the        polynucleotide of embodiment E89, the expression vector or        expression vectors in any one of embodiments E90, E94 or E95,        the host cell of embodiments E91 or E96, or a polypeptide or        fusion protein produced by the method of embodiment E92.    -   E98. A method of treating a disease or disorder comprising        delivering to a subject in need thereof a polypeptide or fusion        protein produced by the method of embodiment E93, wherein said        method comprises administration of the ligand to said subject.    -   E99. The method of embodiment E44, wherein the polypeptide or        fusion protein is delivered to the subject by first        administering the first, second, and third polynucleotides.    -   E100. The method of embodiment E98, wherein the polypeptide or        fusion protein is delivered to the subject by first        administering the expression vector or expression vectors of        embodiments E94 or E95.    -   E101. The method of embodiment E98, wherein said polypeptide or        fusion protein is delivered to the subject by first        administering the host cell of embodiments E91 or E96.    -   E102. A pharmaceutical composition comprising the polypeptide or        fusion protein in any one of embodiments E55 to E88, comprising        the polynucleotide of embodiment E89, comprising the expression        vector or expression vectors in any one of embodiments E90, E94        or E95, or comprising the host cell of embodiments E91 or E96,        and a pharmaceutically acceptable carrier, diluent or excipient.    -   E103. A medicament comprising the polypeptide or fusion protein        in any one of embodiments E55 to E88, comprising the        polynucleotide of embodiment E89, comprising the expression        vector or expression vectors in any one of embodiments E90, E94        or E95, or comprising the host cell of embodiments E91 or E96.    -   E104. Use of the pharmaceutical composition of embodiment E102        or the medicament of embodiment E103, wherein said use is for        the treatment of a disease or disorder.    -   E105. Use of the pharmaceutical composition or the medicament        according to embodiment E104, wherein the disease or disorder is        cancer.    -   E106. An Pseudomonas exotoxin A (PE-A) polypeptide, wherein said        polypeptide comprises a mutation at a position corresponding to        amino acid position E184 in SEQ ID NO:1 (or position E196 in SEQ        ID NO:2) wherein an isoleucine at position E184 (or position 196        in SEQ ID NO:2) is substituted with a different amino acid.    -   E107. The polypeptide of embodiment E106, wherein said        polypeptide does not have PE-A biological activity.    -   E108. A method for assaying the immunogenicity of a mutated form        of Pseudomonas exotoxin A (PE-A), wherein said method comprises:        -   (a) contacting immune cells with a mutated form of PE-A; and        -   (b) assaying immune cell stimulation,    -   wherein said mutated form of PE-A comprises a mutation at a        position corresponding to amino acid position E184 in SEQ ID        NO:1 (or position 196 in SEQ ID NO:2) wherein an isoleucine at        position E184 (or position 196 in SEQ ID NO:2) is substituted        with a different amino acid, and wherein said mutated form of        PE-A also comprises one or more additional amino acid        substitutions compared to a wild-type form of PE-A.    -   E109. The method of embodiment E108, wherein said immune cells        are human immune cells.    -   E110. The method of embodiment E109, wherein said immune cells        are human T-cells, cells of a human T-cell lineage, human        B-cells, or cells of a human B-cell lineage.    -   E111. The method of embodiment E47, wherein the ligand is a        compound having Formula I, or a pharmaceutically acceptable salt        thereof.    -   E112. The method of embodiment E47, wherein the ligand is a        compound having Formula II, or a pharmaceutically acceptable        salt thereof.    -   E113. The method of embodiment E47, wherein the ligand is a        compound having Formula III, or a pharmaceutically acceptable        salt thereof.    -   E114. The method of embodiment E47, wherein the ligand is a        compound of Table 3, or a pharmaceutically acceptable salt        thereof.    -   E115. The method of embodiment E47, wherein the ligand is a        compound having Formula III, wherein:        -   A is:

-   -   -   B is:

-   -   -   R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f), R^(3g),            R^(3h), R^(3i) and R^(3j) are independently selected from            hydrogen, halo, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy;        -   R¹ is (C₁-C₆)alkyl, hydroxy(C₁-C₄)alkyl, or (C₂-C₄)alkenyl;            and        -   R² is optionally substituted (C₁-C₆)alkyl, or a            pharmaceutically acceptable salt thereof.

    -   E116. The method of embodiment E47, wherein the ligand is a        compound selected from the group consisting of:

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-methoxy-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-chloro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-ethyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methoxy-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-chloro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-difluoro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,6-dichloro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,4-dimethoxy-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-difluoro-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(4-methyl-benzo[1,3]dioxole-5-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-1-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(naphthalene-2-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(thiophene-2-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2,5-dimethyl-furan-3-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(2-chloro-pyridine-3-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(6-chloro-pyridine-3-carbonyl)-hydrazide;

-   (R)-3,5-Dimethyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;

-   (R)-3,5-Dimethoxy-4-methyl-benzoic acid    N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;    and

-   (R)-3,5-Dimethyl-benzoic acid    N′-(4-ethyl-benzoyl)-N-(1-phenethyl-but-3-enyl)-hydrazide,    -   or a pharmaceutically acceptable salt thereof.

EXAMPLES Example 1: T Cell Epitope Mapping of PE

Peptides spanning the sequence of an approximately 38 kD form ofPseudomonas exotoxin A protein (“PE38”) were analyzed for the presenceof immunogenic CD4+ T cell epitopes using EPISCREEN™ T cell epitopemapping analysis (Antitope Ltd, Cambridge, UK).

EPISCREEN™ is a proprietary technology commercially available throughAntitope Ltd, Cambridge, UK, to map T cell epitopes within a proteinsequence to determine potential for immunogenicity (based on the numberand potency of T cell epitopes within a sequence). EPISCREEN™ T cellepitope mapping typically uses CD8+ T cell depleted PBMCs from a minimumof 50 HLA-typed donors (selected to represent the human population ofinterest). Typically, 15mer peptides with 12 amino acid overlapsspanning a protein sequence are analyzed in a large number of replicatecultures for in vitro CD4+ T cell stimulation by 3H TdR incorporation.CD4+ T cell stimulation is often detected in two or three adjacent andoverlapping peptides since the core 9mer that binds the MHC class IIbinding groove will be present in more than one peptide sequence.Following identification of peptides that stimulate CD4+ T cells invitro, in silico technology can be used to design epitope-depleted(deimmunized) variants by determining the precise location of core 9mersequences and the location of key MHC class II anchor residues.

A total of 120 overlapping 15mer peptides spanning the entire PE38sequence (SEQ ID NO:2), including 4 peptides covering a null mutationand 4 peptides spanning an N-terminal linker sequence (SEQ ID NO:3) weretested against a cohort of 52 healthy donors. CD4+ T cell responsesagainst individual peptides were measured using proliferation assays(3H-thymidine incorporation). The proliferation assay data was used tocompile a T cell epitope map of the PE38 sequence and six T cellepitopes were identified.

EPISCREEN™ Donor Selection

Peripheral blood mononuclear cells (PBMC) were isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) obtainedfrom the UK National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK) and according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee. PBMC were isolated from buffycoats by LYMPHOPREP™ (Axis-Shield UK, Dundee, Scotland) densitycentrifugation. (LYMPHOPREP™ is a ready-made, sterile and endotoxintested solution for the isolation of human mononuclear cells from blood.See, Axis-Shield, package insert for LYMPHOPREP™ density gradient mediaNo. 619. March 03. Div.—1114740.) CD8+ T cells were depleted using CD8+ROSETTESEP™ (STEMCELL™ Technologies Inc, Manchester, UK) to remove CD8+cells from the isolated mononuclear cells. See e.g., StemCellTechnologies Inc., ROSETTESEP™ procedure for Human CD8+ T CellEnrichment Cocktail (Catalog #15023/15063; Procedure version 1.3.0,“#28572 (May 2011)).

HLA allotypes of donors were characterized using the Biotest HLA SSP-PCRtissue-typing kit (Biotest, Solihull, UK, catalogue number 826215). Tcell responses to a reproducibility control neo-antigen were alsodetermined using Imject maricutlure keyhole limpet haemocyanin (KLH)(Pierce (Perbio Science UK, Ltd)), Cramlington, UK, catalogue number77600) with the KLH diluted to a final concentration of 100 μg/ml. PBMCwere then frozen and stored in liquid nitrogen until required.

A cohort of 52 donors was selected to best represent the number andfrequency of HLA-DR allotypes expressed in the world population.Analysis of the allotypes expressed in the cohort against thoseexpressed in the world population revealed that coverage of >80% wasachieved and that all major HLA-DR alleles (individual allotypes with afrequency >5% expressed in the world population) were well represented.Details of individual donor haplotypes and a comparison of the frequencyof MHC class II haplotypes expressed in the world population and thesample population are shown in Table 7 and FIG. 2, respectively.

Table 7.

Donor details and haplotypes. Donor responses (SI) to KLH are shown fortwo independent proliferation assays. Test 1 was performed using KLH onfreshly isolated PBMC and IEX01 is the KLH re-test performed in thecurrent study on PBMC recovered from liquid nitrogen storage asindicated above. Responses that did not produce the same result (i.e.positive including borderline SI>1.90 p<0.05 or negative) in both testsare highlighted in grey (i.e., donors 3, 7, 9, 33 and 44).

EPISCREEN™ Analysis: Proliferation Assay

PBMC from each donor were thawed, counted and viability was assessed.Cells were revived in room temperature AIM V® culture medium(Invitrogen, Paisley, UK) before adjusting the cell density to 2-3×10⁶PBMC/ml (proliferation cell stock). Peptides were synthesized on a 1-3mg scale with free N-terminal amine and C-terminal carboxylic acid.Peptides were dissolved in DMSO to a concentration of 10 mM and peptideculture stocks prepared by diluting into AIM V® culture medium to afinal concentration of 5 μM in the well. For each peptide and eachdonor, sextuplicate cultures were established in a flat bottomed 96 wellplate. Both positive and negative control cultures were also tested insextuplicate. For each donor, three control antigen/peptides (KLHprotein and peptides derived from Influenza A and Epstein Barr viruses)were also included.

Cultures were incubated for a total of 6 days before adding 0.75 μCi3[H]-thymidine (PERKIN ELMER®, Beaconsfield, UK) to each well. Cultureswere incubated for a further 18 hours before harvesting onto filter matsusing a TOMTEC MACH® III cell harvester (TOMTEC®, Hamden, Conn., USA).Counts per minute (cpm) for each well were determined by Meltilex™(PERKIN ELMER®) scintillation counting on a Microplate Beta Counter(PERKIN ELMER®) in paralux, low background counting mode.

EPISCREEN™ Data Analysis

For proliferation assays, an empirical threshold of a stimulation index(SI) equal to or greater than 2 (SI≧2.00) has been previouslyestablished whereby samples inducing proliferative responses above thisthreshold are deemed positive (where included, borderline SI≧1.90 arehighlighted). Extensive assay development and previous studies haveshown that this is the minimum signal to noise threshold allowingmaximum sensitivity without detecting large numbers of false positiveresponses. Positive responses are defined by the following statisticaland empirical thresholds:

-   -   1. Significance (p<0.05) of the response by comparing cpm of        test wells against medium control wells using unpaired two        sample Student's t-test.    -   2. Stimulation index greater than 2.00 (SI≧2.00), where SI=mean        cpm of test wells/mean cpm medium control wells. Data presented        in this way is indicated as SI≧2.00, p<0.05.

In addition, intra-assay variation was assessed by calculating thecoefficient of variance and standard deviation (SD) of the raw data fromreplicate cultures.

Proliferation assays were set up in sextuplicate cultures (“non-adjusteddata”). To ensure that intra-assay variability was low, the data wasalso analyzed after removing the maximum and minimum cpm values(“adjusted data”) and the SI of donor responses was compared using bothdata sets.

T cell epitopes were identified by calculating the average frequency ofpositive responses (defined above) to all peptides in the study plusstandard deviation (SD) to give a background response threshold. Anypeptide that induced a frequency of positive proliferation responsesabove this threshold in both the adjusted and non-adjusted data wasconsidered to contain an immunogenic T cell epitope (and, thus,potentially represents an immunogenicity inducing epitope which couldgive rise to immunogenic responses in vivo).

In Silico Analysis of Peptides

The sequences of peptides that were positive in the proliferation assaywere analyzed using Antitope's predictive iTOPE™ software (Perry et al.2008). This software predicts favorable interactions between amino acidside chains of the peptide and specific binding pockets within the MHCclass II binding groove. Analysis of the peptide sequences using iTOPE™was performed with overlapping 9mers spanning the peptides which weretested against each of the 34 MHC class II alleles. Each 9mer was scoredbased on the potential ‘fit’ and interactions with the MHC class IImolecules. 9mers that produced a high mean binding score were identifiedand, from the T cell proliferation data, 9mers which were considered ascritical to T cell responses (“core 9mers”) were highlighted. iTOPE™analysis was then repeated with a range of amino acid changes in thecore 9mers in order to determine preferred amino acid substitutions foruse in deimmunization.

Results and Discussion

A total of 120 peptides were synthesized spanning the entire PE38sequence. The peptides were designed as 15mers to span the sequence inoverlapping increments of 12 amino acids. These peptides were thentested for the presence of CD4+ T cell epitopes by EPISCREEN™ T cellepitope mapping analysis. Positive T cell responses were defined bydonors that produced a significant (p<0.05) response with a SI≧2.00 toany given peptide (SI≧2.00, p<0.05). T cell epitopes were identified bycalculating the average frequency of the positive responses to allpeptides in the study plus SD (termed ‘background response threshold’).This was calculated to be 10.8% in the raw ‘non-adjusted’ data and 10.7%in the adjusted data (where maximum and minimum values were removed andthe mean cpm calculated on the remaining four wells). Thus, peptidescontaining a T cell epitope induced positive T cell proliferationresponses (SI≧2.00, p<0.05) in ≧6 donors in the non-adjusted andadjusted data sets. Inter-assay variability was assessed using KLH as areproducibility control where the frequency of positive T cell responsesagainst KLH were compared in two separate EPISCREEN™ assays (Table 7).The results show that inter-assay variability is within the acceptablerange and consistent with previous studies (≦10%). The frequency of Tcell responses against the two control peptides C3 (EBNA derivedepitope) and C32 (Influenza derived epitope) ranged between 23-31%(non-adjusted) and between 21-29% (adjusted) for the two peptides,respectively (FIG. 3). This is within the typical range observed forthese two peptides in T cell epitope mapping studies.

The output from non-adjusted and adjusted data analysis was examined toensure that intra-assay variability was low and that positive responseswere not the result of spurious proliferation in individual wells. Theresults from each analysis showed, in most cases, only small differencesbetween the methods and donor responses for both non-adjusted andadjusted analysis. Table 10 provides a summary of individual donorresponses to each of the peptides. The proliferation assay data showingthe frequency of positive donor responses to each peptide is shown inFIG. 3. For all peptides that induced a high frequency of positive(SI≧2.00, p<0.05, including borderline responses) T cell proliferationresponses above the background response threshold, additional in silicoanalysis was performed to aid in the identification of the preciselocation of MHC class II core 9mer binding registers (using iTOPE™), andto identify peptides that are homologous to sequences containing T cellepitopes that have been tested in previous EPISCREEN™ T cell epitopemapping assays (using TCED™).

Table 8.

Summary of individual donor responses to PE38 peptides. Positiveresponses (SI≧2.00, p<0.05, including borderline responses) areindicated by the donor number and individual SI are shown in parenthesesnext to the corresponding donor. The background response rate was 10.8%in the non-adjusted data and 10.7% in the adjusted data peptidesinducing positive T cell proliferation above this frequency (positiveresponse in ≧6 donors) contained T cell epitopes (indicated with boldtext; i.e., peptides 50, 52, 53, 65, 67, 68, 81, 82 and 110 (also asindicated in FIGS. 3-6)).

TABLE 8 Donor Responses to PE38 Peptides Peptide ProliferationProliferation Peptide # Non-Adjusted Adjusted Sequence   111(2.25), 19(2.42), 25(3.24), 11(2.34), 19(2.72), GGGGGSGGGGGSPEG35(2.77), 36(2.21) 25(3.33), 35(2.76), (SEQ ID NO: 11) 36(2.10)   219(2.44), 36(1.94) 19(27), 48(2306) GGSGGGGGSPEGGSL (SEQ ID NO: 12)   311(1.98), 16(1.92), 19(2.54) 11(1.99), 19(2.78), GGGGGSPEGGSLAAL38(5.37) (SEQ ID NO: 13)   4 16(2.33), 17(2.02), 19(2.29),16(2.13), 17(2.02), GGSPEGGSLAALTAH 35(2.33) 19(2.46), 35(2.35),(SEQ ID NO: 14) 38(3.71)   5 7(1.97), 10(2.24), 17(2.24),7(2.05), 10(2.46), PEGGSLAALTAHQAC 24(1.90) 17(2.20) (SEQ ID NO: 15)   63(2.00) GSLAALTAHQACHLP (SEQ ID NO: 16)   7 3(2.11), 17(2.39), 24(2.20),3(2.30), 17(2.22), AALTAHQACHLPLET 45(2.11) 24(2.11), 45(1.90)(SEQ ID NO: 17)   8 17(2.23), 45(2.12) 17(2.04) TAHQACHLPLETFTR(SEQ ID NO: 18)   9 — — QACHLPLETFTRHRQ (SEQ ID NO: 19)  10 — —HLPLETFTRHRQPRG (SEQ ID NO: 20)  11 10(2.48), 24(2.13) 7(1.95)LETFTRHRQPRGWEQ (SEQ ID NO: 21)  12 — — FTRHRQPRGWEQLEQ (SEQ ID NO: 22) 13 10(3.20), 24(2.12) 10(7.11), 24(2.12) HRQPRGWEQLEQCGY(SEQ ID NO: 23)  14 10(2.14), 17(2.47), 24(2.06) 1(2.22), 10(2.04),PRGWEQLEQCGYPVQ 17(2.25), 24(2.17), (SEQ ID NO: 24) 51(1.91)  1517(2.04), 24(2.24) 17(2.05), 24(2.21) WEQLEQCGYPVQRLV (SEQ ID NO: 25) 16 — — LEQCGYPVQRLVALY (SEQ ID NO: 26)  17 — — CGYPVQRLVALYLAA(SEQ ID NO: 27)  18 — — PVQRLVALYLAARLS (SEQ ID NO: 28)  19 — —RLVALYLAARLSWNQ (SEQ ID NO: 29)  20 3(2.35), 40(1.96) 3(9.55), 8(2.06),ALYLAARLSWNQVDQ 9(1.90), (SEQ ID NO: 30) 19(1.94), 40(2.07)    2111(1.90) 3(4.22), 8(2.22), LAARLSWNQVDQVIR 11(2.17), 40(1.96)(SEQ ID NO: 31)  22 392.20), 40(2.10) 3(4.32), 6(2.17), RLSWNQVDQVIRNAL19(2.00), 40(2.15) (SEQ ID NO: 32)  23 6(2.24), 19(1.97)3(4.67), 6(2.40), WNQVDQVIRNALASP 19(2.22(, (SEQ ID NO: 33)  24 3(3.08)3(3.99) vDQVIRNALASPGSG (SEQ ID NO: 34)  25 3(1.90), 6(1.95) 6(2.36)VIRNALASPGSGGDL (SEQ ID NO: 35)  26 — — NALASPGSGGDLGEA (SEQ ID NO: 36) 27 — — ASPGSGGDLGEAIRE (SEQ ID NO: 37)  28 24(2.62) 9(1.99), 11(1.92),GSGGDLGEAIREQPE 24(3.47) (SEQ ID NO: 38)  29 4(2.05), 17(2.07), 45(2.18)4(1.91), 17(1.96), GDLGEAIREQPEQAR 24(2.29), 45(2.18) (SEQ ID NO: 39) 30 17(1.94) 8(2.54), 17(2.18), GEAIREQPEQARLAL 24(2.44) (SEQ ID NO: 40) 31 — — IREQPEQARLALTLA (SEQ ID NO: 41)  32 — 31(1.96) QPEQARLALTLAAAE(SEQ ID NO: 42)  33 — — QARLALTLAAAESER (SEQ ID NO: 43)  34 — —LALTLAAAESERFVR (SEQ ID NO: 44)  35 4(1.91), 35(1.91),29(2.42), 35(1.98), TLAAAESERFVRQGT 37(2.18), 42(2.05)36(2.05), 37(2.27), (SEQ ID NO: 45) 42(2.07)  36 3(2.14), 42(1.91)3(2.12), 29(3.91), AAESERFVRQGTGND 35(2.06), 37(2.20) (SEQ ID NO: 46) 37 37(2.34), 42(1.96 13(1.91), 29(3.91), SERFVRQGTGNDEAG35(2.06), 37(2.20) (SEQ ID NO: 47)  38 37(2.20), 42(2.12)29(2.28), 36(1.95) FVRQGTGNDEAGAAS 37(2.20), 42(2.06) (SEQ ID NO: 48) 39 42(1.90) — QGTGNDEAGAASGPA (SEQ ID NO: 49)  40 1(2.21) 1(2.08)GNDEAGAASGPADSG (SEQ ID NO: 50)  41 1(2.28) 1(2.16) EAGAASGPADSGDAL(SEQ ID NO: 51)  42 — — AASGPADSGDALLER (SEQ ID NO: 52)  43 — —GPADSGDALLERNYP (SEQ ID NO: 53)  44 17(2.08), 22(1.95), 42(2.02)17(2.13), 22(2.00), DSGDALLERNYPTGA 37(1.98) (SEQ ID NO: 54)  45 — —DALLERNYPTGAEFL (SEQ ID NO: 55)  46 31(2.23) 31(1.98) LERNYPTGAEFLGDG(SEQ ID NO: 56)  47 — — NYPTGAEFLGDGGDI (SEQ ID NO: 57)  48 2(2.63) —TGAEFLGDGGDISFS (SEQ ID NO: 58)  49 — — EFLGDGGDISFSTRG (SEQ ID NO: 59) 50 10(2.54), 11(1.93), 19(2.29), 10(2.56), 11(2.33), GDGGDISFSTRGTQN36(2.36), 37(1.92), 39(2.17), 19(2.37), 36(2.37), (SEQ ID NO: 60)42(2.66), 45(1.96) 39(2.13), 42(2.63), 45(1.95), 46(1.93)  5119(2.03), 42(2.25), 45(1.93) 292.57), 11(2.06), DGISFSTRGTQNWTV19(1.97), 42(2.20), (SEQ ID NO: 61) 45(1.90)  523(7.10), 11(2.76), 16(2.41), 2(1.95), 3(3.19), SFSTRGTQNWTVERL19(2.36), 42(1.97), 44(1.92) 11(3.01), 16(2.58), (SEQ ID NO: 62)19(2.45), 42(2.02), 44(2.05)  53 2(2.13), 395.19), 11(1.98),2(2.27), 3(4.50), TRGTQNWTVERLLQA 16(2.12), 19(2.19), 27(2.09),11(2.01), 16(1.94), (SEQ ID NO: 63) 45(1.92) 19(2.10), 27(2.46)  54 —3(1.90), 11(1.95), TQNWTVERLLQAHRQ 16(1.92) (SEQ ID NO: 64)  55 3(1.98)— WTVERLLQAHRQLEE (SEQ ID NO: 65)  56 — — ERLLQAHRQLEERGY(SEQ ID NO: 66)  57 — — LQAHRQLEERGYVFV (SEQ ID NO: 67)  5810(2.67), 11(2.90) 9(1.99), 10(2.55), HRQLEERGYVFVGYH 11(3.46), 4(1.90)(SEQ ID NO: 68)  59 9(2.27), 37(2.56), 42(2.70) 9(2.38), 11(2.15),LEERGYVFVGYHGTF 37(2.71), 42(3.01) (SEQ ID NO: 69)  60 — 16(2.09)RGYVFVGYHGTFLEA (SEQ ID NO: 70)  61 — 11(2.07) VFVGYHGTFLEAAQS(SEQ ID NO: 71)  62 — — GYHGTFLEAAQSIVF (SEQ ID NO: 72)  63 3(2.88)11(1.97), 16(2.02) GTFLEAAQSIVFGGV (SEQ ID NO: 73)  64 — —LEAAQSIVFGGVRAR (SEQ ID NO: 74)  65 2(2.17), 4(1.94), 14(3.63),2(2.30), 4(1.93), AQSIVFGGVRARSQD 17(2.19), 18(2.46), 36(2.06),11(2.10), 14(3.65), (SEQ ID NO: 75) 39(1.97), 51(7.91)18(2.31), 36(2.09), 39(2.04), 51(6.71)  66 18(1.95), 19(1.90), 36(1.94),19(2.02), 36(1.98), IVFGGVRARSQDLDA 51(10.38) 47(1.91), 51(9.41)(SEQ ID NO: 76)  67 6(2.07), 14(2.62), 16(2.21), 14(2.68), 16(2.55)GGVRARSQDLDAIWR 1792.11), 18(2.60), 42(1.95), 18(2.42), 19(2.06),(SEQ ID NO: 77) 47(1.93), 51(6.83) 3891.95), 47(1.95), 51(5.22)  682(2.07), 14(2.24), 18(2.70) 2(2.12), 11(2.11), RARSQDLDAIWRGFY3891.94), 3992.050, 42(2.10), 14(2.04), 16(2.000, (SEQ ID NO: 78)51(3.69) 19(2.06), 38(2.15), 39(2.17), 51(3.62)  6931(1.95), 42(1.99), 51(2.47) 31(1.93), 51(2.19) SQDLDAIWRGFYIAG(SEQ ID NO: 79)  70 24(2.22) LDAIWRGFYIAGDPA (SEQ ID NO: 80)  71 —IWRGFYIAGDPALAY (SEQ ID NO: 81)  72 — — GFYIAGDPALAYGYA (SEQ ID NO: 82) 73 6(1.91), 14(2.70), 17(2.13), 11(2.02), 14(2.77), IAGDPALAYGYAQDQ39(1.98) 17(1.94), 39(2.01) (SEQ ID NO: 83)  746(1.99), 14(2.77), 38(1.99), 14(2.89), 16(2.01), DPALAYGYAQDQEPD39(2.25), 42(1.90) 39(2.27) (SEQ ID NO: 84)  756(2.22), 14(2.27), 17(1.93), 14(2.24), 16(2.26), LAYGYAQDQEPDARG39(2.05) 39(2.07) (SEQ ID NO: 85)  76 14(2.20), 17(1.98)14(2.40), 39(1.94) GYAQDQEPDARGRIR (SEQ ID NO: 86)  77 — 38(1.90)QDQEPDARGRIRNGA (SEQ ID NO: 87)  78 — — EPDARGRIRNGALLR (SEQ ID NO: 88) 79 — 24(1.91) ARGRIRNGALLRVYV (SEQ ID NO: 89)  809(2.89), 11(2.85), 19(2.18), 9(2.84), 19(2.03), RIRNGALLRVYVPRS36(2.63), 42(2.23), 45(2.23) 36(2.59), 42(2.29) (SEQ ID NO: 90) 45(2.20) 81 1(2.08), 8(1.96), 9(2.05), 1(2.13), 9(2.05), NGALLRVYVPRSSLP11(3.22), 13(2.09), 19(2.09), 13(2.10), 19(2.10), (SEQ ID NO: 91)36(2.21), 42(2.31), 45(2.13), 36(2.15), 42(2.31), 49(2.07)45(1.94), 49(2.00), 51(2.25)  82 9(1.93), 10(2.01), 11(2.41),9(1.90), 10(2.00), LLRVYVPRSSLPGFY 13(2.09), 16(2.11), 19(2.01),13(2.21), 16(2.23), (SEQ ID NO: 92) 36(2.25), 45(1.97), 49(2.44)19(1.98), 36(2.28) 45(1.98), 49(2.37)  83 33(2.02), 42(2.14), 46(1.90),33(1.97), 42(2.14), VYVPRSSLPGFYRTG 49(1.92) 49(1.95) (SEQ ID NO: 93) 84 11(1.93) — PRSSLPGFYRTGLTL   (SEQ ID NO: 94)  85 — — SLPGFYRTGLTLAAP(SEQ ID NO: 95)  86 — — GFYRTGLTLAAPEAA (SEQ ID NO: 96)  87 — —RTGLTLAAPEAAGEV (SEQ ID NO: 97)  88 9(2.59), 11(3.03), 42(2.03),9(2.47), 42(2.01) LTLAAPEAAGEVERL (SEQ ID NO: 98)  899(1.91), 11(4.31), 42(2.34), 11(2.05), 13(2.28), AAPEAAGEVERLIGH49(2.09), 51(5.22) 42(2.16), 51(6.48) (SEQ ID NO: 99)  9011(2.59), 14(2.07), 49(2.12), 14(2.11), 49(2.11), EAAGEVERLIGHPLP51(7.78) 51(6.45) (SEQ ID NO: 100)  91 11(4.15), 42(1.99), 51(4.84)42(2.06), 51(4.07) GEVERLIGHPLPLRL (SEQ ID NO: 101)  9211(2.19), 49(1.99) — ERLIGHPLPLRLDAI (SEQ ID NO: 102)  93 — —IGHPLPLRLDAITGP (SEQ ID NO: 103)  94 — — PLPLRLDAITGPEEE(SEQ ID NO: 104)  95 3(2.10), 7(1.91), 18(1.90), 3(2.00), 7(1.95),LRLDAITGPEEEGGR 19(2.07), 35(2.17) 19(2.04), 45(2.03) (SEQ ID NO: 105) 96 3(2.62), 13(2.19), 16(2.18), 3(2.34), 13(2.46), DAITGPEEEGGRLET39(1.96) 16(2.24), 31(1.93) (SEQ ID NO: 106) 39(2.10)  9713(2.29), 16(2.32), 19(2.20) 13(2.48), 16(2.44), TGPEEEGGRLETILG35(2.43), 45(2.13) 19(2.31), 45(2.16) (SEQ ID NO: 107)  9811(1.92), 13(1.97), 16(2.26), 11(2.26), 13(2.04), EEEGGRLETILGWPL35(1.91), 50(1.98) 16(2.33) (SEQ ID NO: 108)  99 35(2.33) —GGRLETILGWPLAER (SEQ ID NO: 109) 100 35(2.20) — LETILGWPLAERTVV(SEQ ID NO: 110) 101 — — ILGWPLAERTVVIPS (SEQ ID NO: 111) 102 27(1.93) —WPLAERTIVVIPSAIP (SEQ ID NO: 112) 103 AERTVVIPSAIPTDP (SEQ ID NO: 113)104 3(2.40), 1(2.20), 22(1.98), 3(2.17), 13(2.05), TVVIPSAIPTDPRNV49(1.91) 16(2.15) (SEQ ID NO: 114) 105 16(2.43), 22(1.96), 45(1.97)16(2.30), 45(1.95),  IPSAIPTDPRNVGGD 49(1.96) (SEQ ID NO: 115) 10616(2.02), 19(2.02) 16(1.95), 19(1.90) AIPTDPRNVGGDLDP (SEQ ID NO: 116)107 19(2.00), 27(2.06) 19(1.93), 27(1.99) TDPRNVGGDLDPSSI(SEQ ID NO: 117) 108 — — RNVGGDLDPSSIPDK (SEQ ID NO: 118) 109 — —GGDLDPSSIPDKEQA (SEQ ID NO: 119) 110 8(2.07), 9(2.35), 11(2.27),9(2.46), 10(2.04), LDPSSIPDKEQAISA 13(2.13), 16(1.91), 19(3.00)13(2.11), 19(1.99), (SEQ ID NO: 120) 35(1.90) 35(1.94), 38(1.95),50(2.01) 111 3(2.29), 8(2.20), 9(1.93), 3(2.33), 8(2.74),SSIPDKEQAISALPD 11(2.08), 16(2.19), 19(2.60) 9(2.02), 13(1.989)(SEQ ID NO: 121) 112 11(2.47), 16(3.07), 19(2.61) 3(2.33), 8(2.74),PDKEQAISALPDYAS 9(2.02), 13(1.98) (SEQ ID NO: 122) 1133(2.07), 11(2.61), 16(2.44), 3(2.04), 11(1.93), EQAISALPDYASQPG 19(2.62)45(1.90) (SEQ ID NO: 123) 114 19(2.04) — ISALPDYASQPGKPP(SEQ ID NO: 124) 115 16(1.99) — LPDYASQPGKPPRED (SEQ ID NO: 125) 116 — —YASQPGKPPREDLK (SEQ ID NO: 126) 117 — — ITGPEEEGGRLDTIL (SEQ ID NO: 127)118 9(2.04), 11(2.26), 16(2.12), 9(2.27) PEEEGGRLDTILGWP 39(1.93), 5(SEQ ID NO: 128) 119 16(2.11), 39(2.13) 14(1.90), 38(1.96),EGGRLDTILGWPLAE 39(2.10) (SEQ ID NO: 129) 120 11(2.13), 39(2.05)39(2.07) RLDTILGWPLAERTV (SEQ ID NO: 130)

T Cell Epitope Map

Epitopes 1 and 2—

Peptides 50, 52 and 53 induced a high number of positive T cellproliferation responses in the study cohort (Table 8 and FIG. 3).Peptide 50 showed the highest number of positive responses with 15.38%donors responding in the non-adjusted dataset, and 15.38% in theadjusted data set, (SI≧2.00, p<0.05). From in silico analysis, theproposed core 9mer in this region is ISFSTRGTQ (SEQ ID NO:5). Peptides52 and 53 induced lower frequencies of response with 11.54% and 13.46%positive donor responses in the non-adjusted dataset, and 13.46% and11.54% in the adjusted datasets, respectively. A core 9mer wasidentified in peptide 50 but was only partially present in peptides 52and 53 suggesting that these peptides must contain a different T cellepitope. In silico analysis of peptides 52 and 53 did not identify anycore HLA-DR restricted 9mers so it is likely that the positive T cellresponses seen are due to a HLA-DQ restricted T cell epitope.

The magnitude of T cell proliferation responses can provide anindication as to the T cell precursor frequency. In general, peptidesthat induce high frequency (of positive responses in the study cohort)and high magnitude T cell proliferation responses are a characteristicof ‘recall-like’ T cell responses in which the T cell pre-cursorfrequency is high. In contrast, naive T cell responses are generallycharacterized by low magnitude T cell proliferation responses (with lowT cell precursor frequencies). Peptides 52 and 53 induced moderatelyhigh magnitude T cell proliferation responses where the mean SI forpositive (SI≧2.00, p<0.05) T cell responses in the non-adjusted andadjusted data sets were 3.09-2.89 (peptide 52) and 2.51-2.55 (peptide53) (Table 9). Thus these peptides may induce T cell responses in clonesthat are present in high frequencies in healthy individuals and may beindicative of a memory T cell response. Peptide 50 induced lowermagnitude T cell proliferation responses where the mean SI were 2.23 and2.28 in the non-adjusted and adjusted datasets respectively suggestingthat this peptide may induce a naïve T cell response (Table 9).

Epitopes 3 and 4

A cluster of T cell responses were observed around peptides 65-68 andthe subsequent analysis revealed the presence of two T cell epitopes inthis region. Peptide 65 stimulated positive T cell proliferationresponses in 15.38% of the study cohort for both non-adjusted andadjusted datasets (Table 8 and FIG. 3) (SI≧2.00, p<0.05). The positiveresponses were high magnitude (mean SI of positive responses ranged from3.04-2.89 in the non-adjusted and adjusted data sets) suggesting thatthe T cell precursor frequency in healthy donors against this epitope ishigh (Table 9). In silico analysis revealed a potential core 9mercomprising IVFGGVRAR (FIG. 5; SEQ ID NO:7). Peptides 67 and 68 inducedfrequencies of response with 15.38% and 13.46% positive donor responsesin the non-adjusted dataset, and 13.46% and 15.38% in the adjusteddatasets, respectively. In silico analysis of these peptides did notidentify any core HLA-DR 9mers so it is likely that the positive T cellresponses seen are due to a HLA-DQ restricted T cell epitope.

Epitope 5

Peptides 81 and 82 stimulated a number of T cell responses in the studycohort (Table 8 and FIG. 3). Peptide 81 had the highest frequency ofresponse of all the peptides tested with a frequency of positiveresponses of 19.23% in the non-adjusted and 17.31% in the adjusted dataset. For peptide 82, the frequency of positive response was 17.31% and15.38% in the non-adjusted and adjusted data sets respectively. Thepositive responses were relatively low in magnitude (mean SI of positiveresponses ranged from 2.12 to 2.22 in the non-adjusted and adjusted datasets) suggesting that the T cell precursor frequency in healthy donorsagainst this epitope is relatively low (Table 9). Adjacent peptide 80induced a sub-threshold response. In silico analysis of peptides 81 and82 suggested a core 9mer of LRVYVPRSS (FIG. 6; SEQ ID NO:9).

Epitope 6

Peptide 110 induced positive T cell responses in 13.46% of the studycohort in non-adjusted and 13.46% in adjusted datasets (Table 8 and FIG.3). The magnitude of positive proliferation responses was low with amean SI of 2.23 for the non-adjusted dataset and 2.07 for the adjusteddataset (Table 9). There was also a sub-threshold response to peptide111. In silico analysis of the peptides sequence revealed a core 9mer,IPDKEQAIS (FIG. 7; SEQ ID NO: 10) which, in addition to peptide 110, wasalso present in peptide 111.

Table 9.

Summary of magnitude (mean SI and standard deviation) and frequency (%donor response) of positive T cell proliferation responses againstpeptides containing T cell epitopes for PE38. The position of p1 inpotential core 9mers are shown as underlined/bolded text (as predictedby iTOPE™) in peptides 50, 65, 81, 82 and 110.

TABLE 9 Magnitude and Frequency of Donor Responses Mean Response (±SD)Mean Frequency Non- (±SD) Peptide Non- Adjusted Adjusted PeptideSequence Adjusted Adjusted Data Data  50 GDGGD I SFSTRGTQN 15.38% 15.38%2.23 ± 0.28 2.28 ± 0.26 (SEQ ID NO: 60)  52 SFSTRGTQNWTVERL 11.54%13.46% 3.09 ± 1.99 2.89 ± 1.50 (SEQ ID NO: 62)  53 TRGTQNWTVERLLQA13.46% 11.54% 2.51 ± 1.18 2.55 ± 0.98 (SEQ ID NO: 63)  65 AQS IVFGGVRARSQD 15.38% 15.38% 3.04 ± 2.04 2.89 ± 1.64 (SEQ ID NO: 75)  67GGVRARSQDLDAIWR 15.38% 13.46% 2.79 ± 1.65 2.69 ± 1.15 (SEQ ID NO: 77) 68 RARSQDLDAIWRGFY 13.46% 15.38% 2.40 ± 0.62 2.28 ± 0.54(SEQ ID NO: 78)  81 NGAL L RVYVPRSSLP 19.23% 17.31% 2.22 ± 0.36 2.12 ±0.12 (SEQ ID NO: 91)  82 L L RVYVPRSSLPGFY 17.31% 15.38% 2.14 ± 0.192.12 ± 0.17 (SEQ ID NO: 92) 110 LDPSS I PDKEQAISA 13.46% 13.46% 2.23 ±0.38 2.07 ± 0.18 (SEQ ID NO: 120)

HLA Analysis

Analysis of the responding donor haplotypes was performed whereby anassociation between MHC class II allotype and a response to a particularpeptide was considered possible if the frequency of the allotype withinthe responding population was double the frequency observed in the studycohort. This analysis was only carried out for peptides that inducedpositive responses above the background response rate in the adjusteddata in the study cohort and was also restricted to allotypes expressedat higher frequencies (>5%) in the study population.

Analysis of responding donor allotypes (Table 10 and FIG. 8) revealedthat there was a possible association between T cell responses topeptides 81, and 82 and MHC class II allotype HLA DRB1*07 which wasexpressed at twice the percentage of positively responding donorscompared to the study population. Peptide 53 also had a possibleassociation with DRB1*11, and peptides 82 and 110 showed possibleassociations with DRB1*15. It should be noted that further studies (suchas MHC class II binding analysis) would be required to show conclusivelythat responses to the T cell epitope are associated with these allotypesas the present analysis was performed on a small group of respondingdonors.

Table 10.

Frequency (expressed as a percentage) of responding donor allotypescompared to the frequency of allotypes expressed in the IEX01 studycohort. An association between MHC class II allotype and a response to aparticular epitope was considered if the frequency of the allotypewithin the responding population was double the frequency observed inthe study population in the adjusted data set. Possible associations areindicated in heavily bordered boxes. The analysis has been restricted toallotypes expressed at higher frequencies (>5%) in the study population.

Results

The results show that six T cell epitopes were present in the PE38sequence. Table 6 Table 11 and FIG. 8 summarize the location of theputative core 9mers in each sequence along with the frequency andmagnitude of T cell responses against each epitope. The T cell epitopesidentified in PE38 were prioritized according to their potency based onthe frequency and magnitude (mean SI) of positive donor responses toeach peptide. However since the responding donor magnitudes were similar(Table 4 Table 9) for most epitopes, the ranking was mainly based onfrequency of positive donor responses (from highest to lowest):

Epitope 5>Epitope 4>Epitope 3>Epitope 1>Epitope 2>Epitope 6

Deimmunization Strategy

The six epitope core 9mer sequences were analyzed by proprietarysoftware (iTOPE™) in order to identify mutations that remove the T cellepitopes by eliminating or significantly reducing binding to MHC classII (Table 11). As part of the strategy as to which residues to mutate,location within the structure was considered, especially whether theresidue is buried, on the surface, or near active sites.

Table 11. Projected mutations to remove MHC class II binding (based uponiTOPE™ and crystal structure data).

TABLE 11 Location of Core 9-mers and Projected Mutations Amino Epi-Acids in Anchor Projected tope Sequence Residues Mutations Notes: 1 I 1A, N, T, P1 (Ile) is partially Q, H surface exposed, therefore S allalternatives should F be possible. S 4 P6 and P9 changes T performequally well, R 6 Q but are less preferred G 7 than P1 changes. T Q 9 N,T 2 G 1 HLA-DQ epitopes have a T strong negative preference Q forpositively charged N 4 K, R residues in key anchor W positions. All fourT 6 K, R mutations are equally V 7 preferred. E R 9 3 I 1 A, N P1 isburied, therefore A V is preferred. F P6 V is partially exposed. G 4 Allmutations should be G tolerated. Preference is V 6 D, M, N D > M > N. R7 A R 9 4 A 1 HLA-DQ epitopes have a R strong negative preference S forpositively charged Q 4 K, R residues in key anchor D positions. All fourL 6 mutations are equally D 7 K, R preferred. A I 9 5 L 1 A P1 is buriedand close in R D, S, A the structure to epitope 3 V P1, thereforechanges are Y 4 limited. For this epitope, V changes at P2 affect P 6binding (D > S > A). R 7 P9 is mostly surface S exposed. Preferred S 9D, E, N, changes are D, E, N, then K, P, T K > P > T. 6 I 1 A, N, T, P1I is partially surface Q, H exposed, therefore all P alternatives shouldbe D possible. K 4 T P4, P6 and P9 changes are E less preferred than P1Q 6 D changes. P6 D ≧ P7 D > A 7 D P4 T. I S 9

Conclusions

EPISCREEN™ T cell epitope mapping of 120 overlapping 15mer peptidesincluding 112 spanning the entire PE38 sequence suggested six novel Tcell epitopes. In silico analysis was used to identify potential core9mers for MHC binding and, together with structural analysis, was usedas a basis for design of changes for re-engineering and deimmunizingPE38 in particular, and PE molecules in general.

Example 2: T Cell Epitope Mapping of Deimmunized/Amino Acid SubstitutedForms of PE

The immunogenicity of amino acid substituted forms of PE can be assessedusing the same procedures as described in Example 1. Accordingly,EPISCREEN™ T cell epitope mapping analysis (Antitope Ltd, Cambridge, UK)analysis permits identification of amino acid substituted epitopes in PEpolypeptides, wherein the introduced amino acid changes result inreduced or undetectable immunogenicity (i.e., for generating deimmunizedforms of PE) as compared to epitopes in corresponding forms of non-aminoacid substituted PE polypeptides.

EPISCREEN™ is a proprietary technology commercially available throughAntitope Ltd, Cambridge, UK, to map T cell epitopes within a proteinsequence to determine potential for immunogenicity (based on the numberand potency of T cell epitopes within a sequence). EPISCREEN™ T cellepitope mapping typically uses CD8+ T cell depleted PBMCs from a minimumof 50 HLA-typed donors (selected to represent the human population ofinterest). Typically, 15mer peptides with 12 amino acid overlapsspanning a protein sequence are analyzed in a large number of replicatecultures for in vitro CD4+ T cell stimulation by 3H TdR incorporation.CD4+ T cell stimulation is often detected in two or three adjacent andoverlapping peptides since the core 9mer that binds the MHC class IIbinding groove will be present in more than one peptide sequence.Following identification of peptides that stimulate CD4+ T cells invitro, in silico technology can be used to design epitope-depleted(deimmunized) variants by determining the precise location of core 9mersequences and the location of key MHC class II anchor residues.

In this case, amino acid substituted PE peptides are analyzed for thepresence of immunogenic CD4+ T cell epitopes using EPISCREEN™ T cellepitope mapping analysis. For example, amino acid substituted 15merpeptides (compared to non-substituted 15mer peptides corresponding to anon-amino acid substituted form of PE) are tested against a cohort ofhealthy donors. CD4+ T cell responses against individual peptides aremeasured using proliferation assays (3H-thymidine incorporation).Proliferation assay data is used to compile a T cell epitope map ofvarying responses to amino acid substituted forms of PE to determinethose amino acid changes producing reduced or abrogated immunogenicresponses.

EPISCREEN™ Donor Assessments

Peripheral blood mononuclear cells (PBMC) are isolated from healthydonor buffy coats (e.g., from blood drawn within 24 hours). For example,PBMC are isolated from buffy coats using density gradient centrifugationusing LYMPHOPREP™ (Axis-Shield UK, Dundee, Scotland) or a similardensity gradient centrifugation media for the isolation of humanmononuclear cells from blood (such methods, media and products are wellknown and routinely used by those skilled in the art). See e.g.,Axis-Shield, package insert for LYMPHOPREP™ density gradient media No.619. March 03. Div.—1114740.) To remove CD8+ cells from the isolatedmononuclear cells, CD8+ T cells are depleted using CD8+ ROSETTESEP™ kit(STEMCELL™ Technologies Inc, Manchester, UK) or similar CD8+ selectionmethods and techniques (such methods, media and products are well knownand routinely used by those skilled in the art). See e.g., StemCellTechnologies Inc., ROSETTESEP™ procedure for Human CD8+ T CellEnrichment Cocktail (Catalog #15023/15063; Procedure version 1.3.0,“#28572 (May 2011)).

Donors HLA-DR haplotypes are determined using methods or kits well-knownand routinely used by those skilled in the art. For example, DonorsHLA-DR haplotypes are determined using a Biotest HLA SSP-PCRtissue-typing kit (Biotest, Solihull, UK, catalogue number 826215). Tcell responses to a reproducibility control antigen are measured using,for example neo-antigen, using Imject maricutlure keyhole limpethaemocyanin (KLH) (Pierce (Perbio Science UK, Ltd), Cramlington, UK,catalogue number 77600), or other similar control antigen (such antigensand methods are well known and routinely used by those skilled in theart). PBMC are frozen and stored in liquid nitrogen until ready for usein to measuring immunogenicity of amino acid substituted forms of PE.

A cohort of donors are selected to best represent the number andfrequency of HLA-DR allotypes expressed in the world population. It isdesirable that allotypes expressed in the cohort represent a coverageof >80% of all major HLA-DR alleles in the world population (i.e.,individual allotypes with a frequency >5% expressed in the worldpopulation are well represented). Records of individual donor haplotypesand comparison of the frequency of MHC class II haplotypes expressed inthe world population and the sample population are recorded andassessed.

Donor responses (SI) to a control antigen (such as KLH) are assessed bycomparing two independent proliferation assays. Test-1 is performedusing the control antigen (such as KLH) on freshly isolated PBMC andTest-2 is the control antigen re-test performed on PBMC recovered fromliquid nitrogen storage, the latter of which are used in assessingimmunogenicity of amino acid substituted epitopes in PE. Responses thatdo not produce the same result in these two tests (i.e. positiveincluding borderline SI>1.90 p<0.05 or negative) in both tests aredisregarded.

EPISCREEN™ Analysis: Proliferation Assay

PBMC from each donor are thawed, counted and viability is assessed.Cells are revived in room temperature AIM V® Culture Medium(INVITROGEN™, Paisley, UK) before adjusting cell density to 2-3×10⁶PBMC/ml (proliferation cell stock). Peptides are synthesized on a 1-3 mgscale with free N-terminal amine and C-terminal carboxylic acid.Peptides are dissolved in DMSO to a concentration of 10 mM and peptideculture stocks are prepared by diluting into AIM V® Culture Medium to afinal concentration of 5 μM per well. For each peptide and each donor,sextuplicate cultures are established in a flat bottomed 96 well plate.Both positive and negative control cultures are tested in sextuplicate.For each donor, three control antigen/peptides (KLH protein and peptidesderived from Influenza A and Epstein Barr viruses) are also included.

Cultures are incubated for 6 days before adding 0.75 μCi 3[H]-thymidine(PERKIN ELMER®, Beaconsfield, UK) to each well. Cultures are incubated afurther 18 hours before harvesting onto filter mats using a TOMTEC MACH®III cell harvester (TOMTEC®, Hamden, Conn., USA). Counts per minute(cpm) for each well are determined by MELTILEX™ (PERKIN ELMER®)scintillation counting on a Microplate Beta Counter (PERKIN ELMER®) inparalux, low background counting mode.

EPISCREEN™ Data Analysis

In proliferation assays, an empirical threshold of stimulation index(SI) equal to or greater than 2 (SI≧2.00) is considered to represent aninduced proliferative response; samples registering values above thisthreshold are deemed positive (values of SI<2.00 but ≧1.90 areconsidered borderline). Extensive assay development and previous studieshave shown that this is the minimum signal to noise threshold allowingmaximum sensitivity without detecting large numbers of false positiveresponses. Positive responses are defined by the following statisticaland empirical thresholds:

-   -   1. Significance (p<0.05) of the response by comparing cpm of        test wells against medium control wells using unpaired two        sample Student's t-test.    -   2. Stimulation index greater than 2.00 (SI≧2.00), where SI=mean        cpm of test wells/mean cpm medium control wells. Thus, data        presented is indicated as SI≧2.00, p<0.05.

In addition, intra-assay variation is assessed by calculating thecoefficient of variance and standard deviation (SD) of raw data fromreplicate cultures.

Proliferation assays are set up in sextuplicate cultures from which“non-adjusted data” is gathered. To ensure intra-assay variability islow, data is also analyzed after removing maximum and minimum cpm values(to produce “adjusted data”) and the SI of donor responses is comparedusing both data sets.

Reactive T cell epitopes are identified by calculating the averagefrequency of positive responses (defined above) to all peptides in thestudy plus standard deviation (SD) to give a background responsethreshold. Any peptide inducing a frequency of positive proliferationresponses above the threshold in both adjusted and non-adjusted data isconsidered to contain an immunogenic T cell epitope (and, thus,potentially represents an immunogenicity inducing epitope which couldgive rise to immunogenic responses in vivo). Output from non-adjustedand adjusted data is examined to ensure that intra-assay variability islow and that positive responses are not the result of spuriousproliferation in individual wells. An example of this type of analysisis provided in Example 1.

A comparison of corresponding forms of non-amino acid substituted PEimmunogenic epitope responses versus responses obtained with amino acidsubstituted PE peptides is used to assess and predict the effects ofvarious amino acid substitutions in reducing or eliminating theimmunogenicity of PE polypeptides (i.e., for making deimmunized forms ofPE).

Assays for measuring and testing the immunogenicity of amino acidsubstituted forms of PE may also be done as described and exemplified inExample 1 (i.e., via proliferation assays quantitating CD4+ T cellresponses) wherein amino acid substituted forms of PE (i.e.,“deimmunized PE” or “DI-PE”), and/or DI-PE conjugates and fusionproteins (e.g., fusions of DI-PE to antibodies or antigen-bindingfragments thereof) are tested and measured for the presence and potencyof immunogenic responses compared to responses induced by correspondingforms of non-amino acid substituted PE peptides, polypeptides, andfusion or conjugation constructs.

Assays for measuring immunogenicity of amino acid substituted forms ofPE specifically (as indicated above), or PE molecules, generally, mayalso be done according to methods routinely used and well-known to thoseof skill in the art. For example, immunogenicity of amino acidsubstituted forms of PE, in particular, or PE molecules, in general, (asindicated above) may be measured in vivo in non-human primates and/or intransgenic mouse model systems.

Example 3: Measuring Biological Activity of Amino Acid Substituted Formsof PE

Assays for measuring the biological activity of amino acidsubstituted/deimmunized forms of PE, may be done according to methodsroutinely used and well-known to those of skill in the art. Measuredbiological activities of deimmunized (“DI”) forms of PE (“DI-PE”), inparticular, or PE molecules, in general, may include, for example,assays to measure:

-   -   a) general or specific inhibition of protein synthesis (i.e.,        measuring inhibition of synthesis of a specific protein (or        specific proteins) or inhibition of overall (mass) protein        synthesis,    -   b) inhibition of translation elongation factor EF-2 biological        activity;    -   c) induction or catalysis of ADP-ribosylation of EF-2; and    -   d) eukaryotic cell killing activity (cell cytotoxicity).

Assays for Biological Activity: Inhibition of Protein Synthesis

In one example, measurement of inhibition of protein synthesis may bedone via use of in vitro transcription/translation assays (which areroutinely used and well-known to those of skill in the art). Forexample, a cell-free assay may be used to measure DI-PE inducedinhibition of in vitro transcription/translation of a target plasmid(such as, but not limited to, T7-luc). In the case of using a T7-luctranscription/translation assay, the biological activity readout wouldbe chemiluminescent measurement of luciferase activity wherein aminoacid substituted forms of PE are compared to corresponding non-aminoacid substituted forms of PE for ability/inability to inhibittranslation of the luciferase enzyme in vitro. In such assays, the PEpolypeptides being assayed can be introduced via expression fromtemplate DNA (e.g., a PCR product) encoding the toxin-conjugate gene, orby directly introducing quantified amounts of PE proteins. Such assaysmay be used to assess IC50 values* of the various forms of PE tested(*IC50=concentration at which 50% of protein synthesis is inhibitedversus standardized control samples lacking PE).

Some examples of kits and reagents available for in vitrotranscription/translation assays include, but are not limited to:

-   -   TNT® SP6 Coupled Reticulocyte Lysate System (e.g., PROMEGA®        catalog #L4610 (PROMEGA® Corp., Madison, Wis., USA)) allows for        eukaryotic cell-free protein expression in a single-tube, as a        coupled transcription/translation process. More traditional        rabbit reticulocyte lysate translations commonly use RNA        synthesized in vitro from SP6, T3 or T7 RNA polymerase promoters        and require three separate reactions with several steps between        each reaction. The TNT® System bypasses many of these steps by        incorporating transcription directly into the translation mix.        See e.g., PROMEGA® Technical Bulletin # TB126 (Revised        December 2010) which is incorporated by reference herein. See        also, Pelham et al., Eur. J. Biochem. 67, 247-56 (1976); Krieg        et al., (1984) Nucl. Acids Res. 12, 7057-7070 (1984). See also,        U.S. Pat. Nos. 5,324,637; 5,492,817; 5,641,641; and, 5,650,289.    -   TNT® T7 Quick Coupled Transcription/Translation System (e.g.,        PROMEGA® catalog #L1170 (PROMEGA® Corp., Madison, Wis., USA))        further simplifies in vitro transcription/translation reactions        by combining RNA polymerase, nucleotides, salts and Recombinant        RNasin® Ribonuclease Inhibitor with the reticulocyte lysate to        form a single TnT® Quick Master Mix. The TnT® Quick Coupled        Transcription/Translation System may be used with plasmids for        transcription and translation of genes cloned downstream from        either the T7 or SP6 RNA polymerase promoters. The TnT® Quick        System includes a luciferase-encoding control plasmid and        Luciferase Assay Reagent, which can be used in a non-radioactive        assay for rapid (<30 seconds) detection of functionally active        luciferase protein. Starting with either circular plasmid DNA or        PCR-generated DNA, in vitro transcription/translation results        may be obtained in 5-6 hours. See e.g., PROMEGA® Technical        Bulletin # TM045 (Revised May 2011) which is incorporated by        reference herein.    -   STEADY-GLO® Luciferase Assay System (e.g., PROMEGA® catalog        #E2510) (PROMEGA® Corp., Madison, Wis., USA)) allows for        high-throughput quantitation of firefly (Photinus pyralis)        luciferase expression in mammalian cells via batch processing of        96- and 384-well plates. The STEADY-GLO® Luciferase Assay System        provides signal half-lives of over 5 hours in commonly used cell        culture media without prior sample processing. Throughput rates        of several thousand samples per hour may be achieved with high        reproducibility under standard laboratory conditions. See e.g.,        PROMEGA® Technical Bulletin # TM051 (Revised March 2009 &        Revised September 2011) which is incorporated by reference        herein. See also, U.S. Pat. Nos. 5,641,641; 5,650,289;        5,583,024; 5,674,713; ands 5,700,673.

Full protocols for use of such kits are provided by the manufacturerwith each kit. A brief example of a typical experimental procedure mayinclude:

-   -   Assembling kit reagents (except target T7-luc plasmid), plus PE        test samples (using an experimentally determined titration of PE        test samples; e.g., in a range of 0-500 ng DNA per reaction for        PCR templates or using a PE protein titre in a range to be        determined experimentally), in a total volume of 12.5 ul        RNAse-free water in PCR tubes or cell wells on plates.    -   For plasmid DNA: Pre-incubate for required time (e.g. 30-60 min,        time to be determined experimentally) at 30° C. to allow        pre-reaction transcription/translation to occur.    -   For purified protein: No pre-incubation step required.    -   Add target plasmid T7-luc (e.g. 250 ng/reaction, determined        experimentally) and incubate further (e.g. 30-60 min, time to be        determined experimentally) at 30° C.    -   Stop reaction by placing on ice. Increase sample volume to 50 ul        with RNAse-free water.    -   Add luciferase reagent (e.g. SteadyGlo, 50 ul per well) to each        well, incubate according to manufacturer's instructions,        transfer to 96 well black/white plate and read chemiluminescent        signal via chemiluminescence platereader.    -   Compare to ‘zero toxin’ control samples (i.e., no PE present) to        determine the % inhibition of transcription/translation (i.e.,        as a function of inhibition of luciferase activity).    -   Compare inhibition of transcription/translation values of amino        acid substituted/deimmunized forms of PE compared to        corresponding forms of non-amino acid substituted PE.

Comparative protein synthesis inhibition values may show that variousforms of DI-PE exhibit 100% or about 100% of biological activity(inhibition of protein synthesis) compared to corresponding forms ofnon-amino acid substituted PE. Comparative protein synthesis inhibitionvalues may also show that various forms of DI-PE exhibit at least 95%,or at least about 95%, at least 90%, at least about 90%, at least 85%,at least about 85%, at least 80%, at least about 80%, at least 75%, atleast about 75%, at least 70%, at least about 70%, at least 60%, atleast about 60%, at least 50%, or at least about 50% of biologicalactivity compared to corresponding forms of non-amino acid substitutedPE.

Assays for Biological Activity: Cell Cytotoxic Activity

In one example, measurement of cell cytotoxic activity may be done viause of in vitro cell based assays wherein deimmunized PE(DI-PE)-antibody conjugates are assayed in comparison to non-amino acidsubstituted PE-antibody conjugates. The antibody portion of suchconjugates would be antibodies, or antigen-binding fragments thereof,which specifically bind antigens expressed on the cell-surface of celltypes used in such in vitro assays. Cell cytotoxicity may bequantitated, for example, by measuring cell lysis wherein the biologicalreadout is represented by measurement of, for example, based onchemiluminescent (LUMI), fluorometric (FL), and colorimetric (COL)outputs; such as can be practiced using commercially available kitsroutinely used and well-known to those of skill in the art.

Some examples of kits available for measurement and comparison of DI-PEversus non-amino acid substituted PE cell cytotoxicity include, withoutlimitation:

-   -   TOXILIGHT® BioAssay Kit (e.g., Catalog # #LT07-117 (Lonza        Rockland, Inc., Rockland, Me., USA)) is a non-destructive        bioluminescent cytotoxicity assay that quantitatively measures        release of Adenylate Kinase (AK) from damaged mammalian cells        and cell lines in vitro. The assay is based on the        bioluminescent measurement of AK which is present in all cells.        A loss of cell integrity, through damage to the plasma membrane,        results in the leakage of a number of factors from cells        cultured in vitro into the surrounding medium. The measurement        of the release of AK from the cells allows the accurate and        sensitive determination of cytotoxicity and cytolysis. The        reaction involves two steps. The first involves the addition of        ADP as a substrate for AK. In the presence of the enzyme, AK,        the ADP is converted to ATP for assay by bioluminescence. The        bioluminescent part of the assay utilizes the enzyme Luciferase,        which catalyses the formation of light from ATP and luciferin.        By combining these two reactions, the emitted light intensity is        linearly related to the AK concentration and can be measured        using a luminometer or beta counter. See, “TOXILIGHT® BioAssay        Kit: Instructions for Use,” 02007 Lonza Rockland, Inc., which is        incorporated by reference herein. See also, Crouch, et al., J.        Immunol. Methods, 160(1):81-88 (1993); Olsson, T. et al., J.        Appl. Biochem 5, 347-445 (1983); and, Squirrell et al., A        Practical Guide to Industrial Uses of ATP Luminescence in Rapid        Microbiology, p. 107-113 (1997).    -   CYTOTOX-GLO® (e.g., PROMEGA® catalog # G9290 (PROMEGA® Corp.,        Madison, Wis., USA)) is a luminescent cytotoxicity assay that        measures the relative number of dead cells in cell populations.        The assay measures extracellular activity of a distinct        intracellular protease activity (dead-cell protease) when the        protease is released from membrane-compromised cells. A        luminogenic cell-impermeant peptide substrate        (AAF-aminoluciferin) is used to measure dead-cell protease        activity. The liberated aminoluciferin product is measured as        “glow type” luminescence generated by ULTRA-GLO™ Recombinant        Luciferase provided in the assay reagent. The AAF-aminoluciferin        substrate cannot cross the intact membrane of viable cells and        does not generate appreciable signal from the live-cell        population. The amount of luminescence directly correlates with        the percentage of cells undergoing cytotoxic stress. With the        addition of a lysis reagent (provided with the kit), the        CYTOTOX-GLO™ Assay provides a luminescent signal associated with        the total number of cells in each assay well. Viability can be        calculated by subtracting the luminescent dead-cell signal from        the total luminescent value, thus allowing normalization of        assay data to cell number and mitigation of assay interferences.        The cytotoxicity protease biomarker is constitutive and        conserved across cell lines. See e.g., PROMEGA® Technical        Bulletin Nos. TB359 (Revised May 2009 & Revised October 2011)        which is incorporated by reference herein. See also, Niles, A.        et al. (2007) Anal. Biochem., 366, 197-206 (2007) and U.S. Pat.        Nos. 6,602,677 and 7,241,584.    -   CYTOTOX-ONE™ kit (e.g., PROMEGA® catalog # G7891 (PROMEGA®        Corp., Madison, Wis., USA)) allows performance of homogeneous        membrane integrity assays wherein a fluorometric method may be        used to estimate the number of nonviable cells present in        multiwell plates. This assay measures the release of lactate        dehydrogenase (LDH) from cells with damaged membranes. LDH        released into the culture medium is measured with a coupled        enzymatic assay that results in the conversion of resazurin into        a fluorescent resorufin product. The amount of fluorescence        produced is proportional to the number of lysed cells (which may        be monitored using a 96- or 384-well plate formats). The        CYTOTOX-ONE™ Reagent does not damage normal healthy cells.        Therefore, reactions to measure released quantities of LDH can        be performed directly in a homogeneous format in assay wells        containing a mixed population of viable and damaged cells. See        e.g., PROMEGA® Technical Bulletin # TB306 (Revised May 2009)        which is incorporated by reference herein. See also, U.S. Pat.        Nos. 6,982,152 and 7,282,348.    -   CELLTITER GLO® Luminescent Cell Viability Assay (e.g., PROMEGA®        catalog #G7571 (PROMEGA® Corp., Madison, Wis., USA)) provides a        homogeneous method for determining the number of viable cells in        a culture based on quantitation of the amount of ATP present (an        indicator of metabolically active cells). The CELLTITER GLO®        Assay is particularly useful for automated high-throughput        screening (HTS), cell proliferation and cytotoxicity assays. The        homogeneous assay procedure involves adding the single reagent        (CELLTITER GLO® Reagent) directly to cells cultured in        serum-supplemented medium. The assay allows for detection of as        few as 15 cells/well in a 384-well format in 10 minutes after        adding reagent and mixing. The homogeneous “add-mix-measure”        format results in cell lysis and generation of a luminescent        signal proportional to the amount of ATP present (which is        directly proportional to the number of cells present in        culture). The CellTiter-Glo® Assay generates a “glow-type”        luminescent signal, which has a half-life generally greater than        five hours, depending on cell type and medium used. See e.g.,        PROMEGA® Technical Bulletin Nos. TB288 (Revised June 2009 &        Revised August 2011) which are incorporated by reference herein.        See also: U.S. Pat. Nos. 6,602,677; 7,241,584; 7,700,310;        7,083,911; 7,452,663; 7,732,128; 7,741,067; 5,583,024,        5,674,713; and 5,700,673.    -   VIALIGHT® Plus Kit (e.g., Catalog # #LT07-221 (Lonza Rockland,        Inc., Rockland, Me., USA)) may be used for rapid detection of        cytotoxicity in mammalian cells and cell lines in culture via        determination of ATP levels. Any form of cell injury results in        a rapid decrease in cytoplasmic ATP levels. Therefore, the        VIALIGHT® Plus Kit may be used to measure a wide range of        biological activities effecting cell viability. The kit is        formulated for use with a microtitre plate reading luminometer        for assay automation. The assay is based on bioluminescent        measurement of ATP is present in all metabolically active cells.        The bioluminescent method utilizes an enzyme, luciferase, which        catalyses the formation of light from ATP and luciferin        according to the following reaction:

ATP+Luciferin+O2-Luciferase/Mg²⁺->Oxyluciferin+AMP+PPi+CO₂+LIGHT

-   -   The emitted light intensity is linearly related to the ATP        concentration and can be measured using a luminometer or beta        counter. The assay is conducted at ambient temperature (18°        C.−22° C.), the optimal temperature for luciferase enzymes. See,        “VIALIGHT® Plus Kit: Instructions for Use,” ○2007 Lonza        Rockland, Inc, which is incorporated by reference herein.

Full protocols for use of such kits are provided by the manufacturerwith each kit. A brief example of a typical experimental procedure mayinclude:

-   -   Plate cells to test plate (e.g., 96 well plates) in growth        medium.    -   Incubate cells with titrations of amino acid substituted forms        of PE-toxin conjugates (including zero toxin and non-amino acid        substituted PE controls (up to a maximum toxicity point, e.g.        100% cell lysis) for required time (determined experimentally,        e.g. 48-72 hr).    -   Add kit reagents for cytotoxicity measurements as per        manufacturer's instructions.    -   Transfer test samples to 96 well black/white walled plate (as        appropriate) and read reaction signal output.    -   Compare cell cytotoxicity values obtained for        substituted/deimmunized forms of PE versus corresponding        non-amino acid substituted forms of PE.

Comparative cell cytotoxicity values may show that various forms ofDI-PE exhibit 100% or about 100% of biological activity (induction ofcell cytotoxicity) compared to corresponding forms of non-amino acidsubstituted PE. Comparative cell cytotoxicity values may also show thatvarious forms of DI-PE exhibit at least 95%, or at least about 95%, atleast 90%, at least about 90%, at least 85%, at least about 85%, atleast 800, at least about 80%, at least 75%, at least about 75%, atleast 70%, at least about 70%, at least 60%, at least about 60%, atleast 50%, or at least about 50% of biological activity compared tocorresponding forms of non-amino acid substituted PE.

Example 4: Measuring Ability of Deimmunized PE Variants to InhibitProtein Synthesis

Quantitative in vitro transcription/translation (IVTT) assays to assessthe biological activity of deimmunized variants of PE in inhibitingprotein synthesis (i.e., possess wild-type PE biological activity) maybe performed using the TNT® Quick Coupled Transcription/TranslationSystems assay from PROMEGA® Corp. (Madison, Wis., USA). See, PROMEGA®Technical Bulletin # TB126 (Revised December 2010) which is incorporatedby reference herein.

Example 5: Measuring Ability of a PE-IL2 Fusion Protein to InhibitProtein Synthesis in an In Vitro Transcription/Translation (IVTT) Assay

A preliminary experiment was performed to compare the ability of aPE-IL2 fusion protein to inhibit protein synthesis in an in vitrotranscription/translation assay when a commercially available PE-ILfusion protein is translated in vitro following transcription fromeither a circular plasmid expression vector or a linearized plasmidexpression vector. The PE-IL2 expression vector in this experiment isreferred to as “VVN-52431.” A few examples of IL2-PE fusion constructare shown in SEQ ID NO:164, 165 and 166. The aim of this experiment wasto determine if circular or linearized plasmids produced significantlydifferent quantities of PE-IL protein in the PROMEGA® Corp. TNT® QuickCoupled Transcription/Translation Systems assay. A commerciallyavailable T7 Promoter/Luciferase expression vector (PROMEGA® Corp.;hereinafter “T7-Luc DNA”) was used to measure the ability of PE-IL2 toinhibit protein synthesis in vitro.

Based on a pilot IVTT experiment, it was determined that 0.2 μg T7-LucDNA provided optimal RLU (Relative Light Units) in a 90 minute IVTTreaction. In this experiment, VVN-52431 was linearized using therestriction enzyme Fsp-I. Linearized and circular VVN-52431 DNA wereused as templates in the IVTT reactions. Reactions were done intriplicate, using 0.5, 1 and 2 μg of DNA. The T7 control reaction wasperformed using 1 μg DNA. Reactions were analyzed via SDS-PAGE and byLuciferase assay.

Materials:

Item Vendor Lot # Nuclease-free water (1000 ml) Ambion 1105062 TNT T7Quick Coupled T/T system PROMEGA ® 328577 T7 luciferase plasmid DNA(From PROMEGA ® same kit) Fsp I NEB 0571101 Dual Glo ® Luciferase AssayPROMEGA ® 322310 System Ultrapure Water GIBCO 896656 Tris-Glycine SDSSample Buffer, Invitrogen 743995 2x 10x Reducing Agent Invitrogen 897034Criterion Tris HCl 4-15%, 1 mm, Bio-Rad 400059499 12 + 2 well PrecisionPlus Protein Standards, Bio-Rad 310009928 Kaleidoscope 10xTris/Glycine/SDS Buffer Bio-Rad 210007884 Gelcode Blue Safe ProteinStain ThermoFisher LL152043

Item Vendor ID # P20, P200, P1,000 Rainin N/A Water bath LuminometerPower Pac HC Bio-Rad N/A Heat block VWR N/A Microcentrifuge,refrigerated Eppendorf N/A Platform Adjustable Tilt Rocker Labnet N/AThermal cycler MJ Research N/A

Procedure:

Per manufacturer's instructions: Except for the actualtranscription/translation incubation, all handling of the TNT® QuickMaster Mix was performed at 4° C. Unused Master Mix was refrozen as soonas possible after thawing to minimize loss of translational activity.

Restriction Digest:

In PCR tubes, the following were combined:

VVN-52431 was linearized by combining the following:

NF H₂O VVN52431 Fsp I Reaction Rxn (μL) (μL) dig. final Product 1 5 5 (5μg) 0 0.5 μg/μl Circular Vector - No Restriction enzyme added 2 4 5 (5μg) 1 0.5 μg/μl Linearized Vector

Reactions were incubated at 37° C. for 60 min.

Reactions were heat inactivated at 65° C. for 20 min.

IVTT Reactions:

-   -   1. In nuclease-free 1.5 ml eppendorf tubes, the following were        combined according to the chart below:        -   Diluted T7-Luc DNA in NF (nuclease free) water at 1:5. Final            DNA concentration=0.1 μg/μl.        -   Serial diluted unlinearized and linearized VVN-52431 DNA            (0.5 μg/μl) in NF water at 1:5. Final concentration=0.5            μg/μl, 0.1 μg/μl, 0.02 μg/μl.

NF T7 Luc Methi- T7 TNT H₂O DNA VVN52431 Fsp onine rex Rxn (μL) (μL)(μL) I dig. (μL) (μL) 1 7 2 (0.2 μg) 1 40 2 7 2 (1 μg) − 1 40 3 7 2 (1μg) + 1 40 4 5 2 (0.2 μg) 2 (1 μg) − 1 40 5 5 2 (0.2 μg) 2 (0.2 μg) − 140 6 5 2 (0.2 μg) 2 (0.04 μg) − 1 40 7 5 2 (0.2 μg) 2 (1 μg) + 1 40 8 52 (0.2 μg) 2 (0.2 μg) + 1 40 9 5 2 (0.2 μg) 2 (0.04 μg) + 1 40 10 9 1 40

-   -   2. Reactions were incubated at 30° C. for 90 minutes in a water        bath.    -   3. Reactions were analyzed for the synthesis of functional        Luciferase using a standard Luciferase assay.

Luciferase Assay:

-   -   1. Luciferase assay substrate was prepared according to        manufacturer's instructions: Reagent Kit was thawed at room        temperature.        -   Dual-Glo® Luciferase Buffer was transferred into the            Dual-Glo® Luciferase Substrate bottle and shaken slightly to            ensure the substrate dissolved.        -   Dual-Glo® Stop & Glo® substrate was transferred into the            Dual-Glo® Stop & Glo® buffer and mixed well.        -   Rehydrated reagent was aliquoted into 15 ml centrifuge tube            (10 ml/tube) and wrapped with Aluminum foil.        -   Rehydrated reagent was stored at −80° C. until ready for use            (the reagent is good for 6 months).    -   2. 5 μl of reaction end products/well were transferred into a        96-well white plate    -   3. 100 μL of the Luciferase Assay Reagent was dispensed per well        and mixed by pipetting 2-3×.    -   4. RLU's for each well on plate were read within 10 minutes.

Results:

Results are shown in FIG. 9. Circular plasmid expression vector encodingPE-IL2 fusion protein was slightly better at inhibiting Luciferaseprotein synthesis compared to linearized plasmid encoding the same (atall Luciferase vector: VVN-52431 vector ratios). These results alsodemonstrate the ability of to test and compare the biological activityof PE-fusion proteins in inhibiting protein synthesis.

In addition to measuring inhibition of protein synthesis as a measure oflight production catalyzed by Luciferase, quantitative analysis ofinhibition of protein synthesis was also performed by separatingpolypeptide reaction products on SDS-PAGE gels, staining, and assessingamounts of polypeptide produced (data not shown).

Assays such as these may be used to compare the ability of amino acidsubstituted (e.g., deimmunized) forms of PE (alone or as fusionproteins) to retain biological activity (such as inhibition of proteinsynthesis) compared to corresponding non-amino acid substituted forms ofPE (alone or as fusion proteins).

Example 6: In Vitro Transcription/Translation (IVTT) Assay to Measureand Compare Ribosylation Activity of Amino Acid Substituted Variants ofPE

Purpose:

This protocol provides an example of they type of methods which may beused to measure and compare the ribosylation activity (i.e., inhibitionof protein synthesis) of amino acid substituted forms of PE compared tocorresponding non-amino acid substituted forms of PE.

Background:

The IVTT assay measures PE mediated inhibition of in vitrotranscription/translation of a target plasmid, T7-Luc. The level ofinhibition (or lack thereof) is determined by chemiluminescentmeasurement of luciferase activity (i.e., the transcribed and translatedprotein). In this assay, a lowered level of transcription andtranslation (and thereby, lowered levels of chemiluminescent lightoutput) corresponds to increased inhibition of protein synthesis. IVTTcan be performed using template DNA encoding PE, or by directly usingquantified protein. This assay may be used to rank different PE variantsagainst each other and to compare their biological activities tocorresponding non-amino acid substituted forms.

Materials:

Test sample: Vectors comprising either circular plasmids with an SP6promoter or linearized plasmids with a T7 promoter.

Reagents and Materials Vendor Nuclease-free water (1000 ml) Ambion TNTSP6 Quick Coupled T/T system PROMEGA ® SP6 luciferase plasmid DNA (FromPROMEGA ® same kit) RNase-free 1.5 ml microfuge tubes Ambion Dual Glo ®Luciferase Assay System PROMEGA ®

Equipment:

Item Vendor P20, P200, P1,000 Rainin 96 well white plate Costar Waterbath (circulation) Luminometer Microcentrifuge, refrigerated Eppendorf

Reagent Preparation:

Luciferase Preparation:

-   -   1. Thaw reagent Kit on ice or at 4 degrees C.    -   2. Transfer entire Dual-Glo® Luciferase Buffer into Dual-Glo®        Luciferase Substrate bottle and shake bottle slightly to ensure        substrate completely dissolved.    -   3. Transfer entire Dual-Glo® Stop & Glo® substrate into        Dual-Glo® Stop & Glo® buffer and mix well.    -   4. Aliquot rehydrated reagent into 15 ml centrifuge tube (10        ml/tube) and wrap tube with Aluminum foil.    -   5. Store rehydrated reagent in −80° C. Freezer (reagent is good        for 6 months)

Procedure:

Per manufacturer's recommendations: Except for the actualtranscription/translation incubation, all handling of TNTR Quick MasterMix should be done at 4° C. Any unused Master Mix should be refrozen assoon as possible after thawing to minimize loss of translationalactivity. Do not freeze-thaw the Master Mix more than two times.

Plasmid DNA Dilution:

Dilute plasmid DNA and Luc plasmid DNA in nuclease free water to finalconcentration of 0.1 μg/μl.

IVTT Reactions

-   -   1. In 1.5 ml NF (nuclease free) eppendorf tubes, the following        are combined for each test sample:        -   a. 5 μL of NF H₂O;        -   b. 2 μl, 0.1 μg/μl test plasmid (for increased accuracy of            results test a dilution series of samples);        -   c. 1 μl, 1 mM Methionine;        -   d. 40 μL TNT quick master mix;        -   e. Negative control (reaction mix only. NF water 9 μl,            methionine 1 μl, reaction mixture 40 μl;    -   2. Incubate reaction mixes at 30° C. for 15 minutes in water        bath;    -   3. Add 2 μl, 0.1 μg/μl Luc plasmid to each reaction mix;    -   4. Incubate reactions at 30° C. for 90 minutes in water bath;        and    -   5. Transfer all samples onto ice to stop reaction.

Luciferase Assay

-   -   6. Transfer 5 μl of end product/well into 96-well white plates        in triplicate;    -   7. Dispense 100 μL of Luciferase Assay Reagent per well. Mix by        pipetting 2-3×;    -   8. Read entire plate within 10 minutes.

Calculations

-   -   1. Calculate percent inhibition based on relative luminescence        units (RFU) of the test sample divided by the RFU of the LUC        plasmid with no test sample, then subtract the result from 100.    -   2. Calculate the percent of activity of non-amino acid        substituted PE by percent inhibition of the test sample divided        by the percent inhibition of non-amino acid substituted, then        multiply the result by 100.    -   3. If a dilution series of samples is tested, calculate the IC50        (half maximal inhibition concentration) for each sample using        the RFU of the test sample divided by the RFU of the LUC plasmid        alone, then subtract the result from 100. Determine the        concentration which results in 50% inhibition.    -   4. Calculate the percent of non-amino acid substituted PE        inhibition by dividing the IC50 of the non-amino acid        substituted PE by the IC50 of the test sample and multiplying        the result by 100.

Example 7: Ex Vivo Assays to Assess Immunogenicity of Amino AcidSubstituted Forms of PE (I.e. Deimmunized PE) Versus CorrespondingNon-Amino Acid Substituted Forms

The immunogenicity of amino acid substituted forms of PE (alone or asPE-fusion proteins) are assessed using methods well-known and routinelyused by those skilled in the art. For example, ELISA assays are usedwherein serum is assayed ex vivo (following extraction from organisms inwhich amino acid substituted forms of PE, or non-amino acid substitutedPE, (alone or as fusion proteins) are administered) to determine whetheror not antibodies that specifically bind the administered protein areproduced. It is noted that in the case of PE-fusion proteins it isnecessary to use, as the ELISA assay target antigen, not only intactPE-fusion proteins (i.e., amino acid substituted or non-amino acidsubstituted PE), but to also test the PE component and the polypeptidefusion component separately to determine whether or not antibodiesproduced specifically bind the PE portion or the fusion polypeptideportion (e.g., IL2 as used in a previous example of a PE-L2 fusionprotein). Accordingly, it is most desirable to identify amino acidsubstituted forms of PE which do not result in host production ofantibodies that specifically bind modified forms of PE (i.e.,deimmunized forms of PE).

Organisms in which amino acid substituted forms of PE may beadministered (alone or as fusion proteins) include, for example, withoutlimitation: mice (including transgenic mice expressing humanimmunoglobulin genes), rats, rabbits, dogs, goats, sheep, horses, cows(and other bovine species), non-human primates, and humans.

Example 8: In Vitro and In Vivo Assays to Assess Cytotoxicity of AminoAcid Substituted Forms of PE

The cytotoxicity of amino acid substituted forms of PE (alone or asPE-fusion proteins) are assessed using methods well-known and routinelyused by those skilled in the art. For example, the cytotoxic effects ofamino acid substituted forms of PE (alone or as PE-fusion proteins)administered to cells in vitro or organisms in vivo, may be assessedwith reference to cytotoxic (cell killing) effects on target cells,organs, tissues, or tumors against which PE or PE fusion proteins areexpected to produce a cytotoxic effect. For example, the therapeuticallybeneficial cytotoxic effects of amino acid substituted PE-Mesothelinfusions may be assessed by monitoring and measuring reduction orelimination of tumor or cancer cells or tissues (in vitro or in vivo) inresponse to administration of amino acid substituted forms ofPE-Mesothelin versus wild-type PE-Mesothelin fusion.

Organisms in which amino acid substituted forms of PE may beadministered (alone or as fusion proteins) include, for example, withoutlimitation: mice (including transgenic mice expressing humanimmunoglobulin genes), rats, rabbits, dogs, goats, sheep, horses, cows(and other bovine species), non-human primates, and humans.

Examples 9-13

Oligonucleotides referenced in the following examples are listed inTable 12.

Example 9: Generation of cDNAs Encoding Amino Acid Substituted Forms ofPE

The Kozak sequence in vector pET14b (EMD Millipore catalog #69660,Darmstadt, Germany) was modified by introducing a linker made up ofannealed oligonucleotides 5′-CATGGTGGCTCTCCTTCTTAAAGTTAAACAAAATTATTT-3′(SEQ ID NO:239)(OL2216 in Table 12) and

5′-CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAGCCAC-3′ (SEQ ID NO:240)(OL2217 inTable 12) (underlined letters indicate nucleotides changed in the Kozaksequence*) via NcoI and XbaI restriction sites into vector pET14bresulting in a modified Kozak sequence (SEQ ID NO:176) by mutation ofthree nucleotides at positions 587 to 589. The resulting vector wasnamed pET14b-K.

*Kozak sequence=(gcc)gccRccAUGG (SEQ ID NO:286), where R is a purine(i.e., adenine or guanine) three bases upstream of the start codon(AUG), which is followed by another ‘G’. See, Kozak, Nucleic Acids Res.15 (20): 8125-8148 (1987).

Oligonucleotides for generation of genes encoding amino acid substitutedforms of PE are listed as OL2164 to OL2194 and OL2281 to OL2366 in Table12. A wild-type (WT) PE gene (SEQ ID NO:1) was made by gene synthesisand amplified using oligonucleotides:5′-ATTGTCCATATGCCAGAAGGCGGTAGCCTGGC-3′ (SEQ ID NO:215)(OL2154 in Table12) to introduce a NdeI site, and

-   -   5′-ATCCTCGAGTTACTTCAGGTCCTCACGCGGCG-3′ (SEQ ID NO:222)(OL2167 in        Table 12) to introduce a XhoI site. The resulting DNA fragment        was subcloned into pGEMT®-T (PROMEGA® catalogue # A1360,        PROMEGA®, Southampton, UK). Colonies were screened by PCR using        M13 primers OL0001 and OL0002 (Table 12). Subsequently the        wild-type (WT) PE gene was subcloned into pET14b-K using NdeI        and XhoI restriction enzymes (Fermentas catalog # FD0583 and        FD0695, respectively) and resulting in a gene encoding an        N-terminal His6 tag fused to the WT PE sequence. The resulting        vector was named pET14b-K-WT PE.

Oligonucleotides for generation of genes encoding amino acid substitutedforms Genes encoding amino acid substituted forms of PE were generatedusing overlapping PCR with the WT PE gene in pET14b-K as template. Pairsof primers from the oligonucleotides of Table 12 (as noted in the“application” column of Table 12) were annealed to WT PE DNA and theamino acid substituted genes were PCR amplified using terminaloligonucleotides: 5′-ATCTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG-3′ (SEQ IDNO:241)(OL2268 in Table 12) and

-   -   5′-ATCCTCGAGTTACTTCAGGTCCTCACGCGGCG-3′ (SEQ ID NO:216)(OL2161 in        Table 12). PCR fragments were and cloned into pET14b-K using        XbaI and XhoI restriction sites.

TABLE 12 Oligonueleotides Name sequence length application OL  001CGCCAGGGTTTTCCCAGTCAC 24 M13 FOR GAC (SEQ ID NO: 205) OL  002AGCGGATAACAATTTCACACA 24 M13 REV GGA (SEQ ID NO: 206) OL 2043GAAGTGCAGCTGGTGGAG 18 RFB4 VH5′ PCR primer (SEQ ID NO: 207) sequence OL2044 CAGAGCCACCTCCGCCTGAAC 49 RFB4 VH3′ PCR primer CGCCTCCACCTGAGGAGACAsequence GTGACCAG (SEQ ID NO: 208) OL 2045 CAGGCGGAGGTGGCTCTGGC 50RFB4 VK 5′ PCR Primer GGTGGCGGATCGGATATCCA Sequence GATGACCCAG(SEQ ID NO: 209) OL 2046 TTTGATCTCCAGCTTGGTG 19 RFB4 VK 3′ PCR Primer(SEQ ID NO: 210) sequence OL 2047 CCCAGCCGGCCATGGCGGAA 35RFB4 Pull through Primer GTGCAGCTGGTGGAG (FOR)) (SEQ ID NO: 211) OL 2048GGTGCTCGAGTGCGGCCGCCC 41 RFB4 Pull through Primer GTTTGATCTCCAGCTTGGTG(REV) (SEQ ID NO: 212) OL 2097 AACCGCCCGGCCGTTCTTCTC 39IEX02 GroEL/ES REV CGTGTTGCCCGGAAAGCC (SEQ ID NO: 213) OL 2098GGGCCAAAGCTTGTTCTTGTT 36 IEX02 GroEL/ES FOR TGAGTCCACTCATGG(SEQ ID NO: 214) OL 2154 ATTGTCCATATGCCAGAAGGC 32 IEX02 PE38 FOR,GGTAGCCTGGC introducing NdeI (SEQ ID NO: 215) OL 2161ATCCTCGAGTTACTTCAGGTC 32 IEX02 PE38 REV, CTCACGCGGCG introducing XhoI(SEQ ID NO: 216) OL 2162 GGGTGGTCGCCTGGACACTAT 30 IEX02 PE38 NM E229DCCTGGGTTG FOR (SEQ ID NO: 217) OL 2163 CAACCCAGGATAGTGTCCAG 30IEX02 PE38 NM E229D GCGACCACCC REV (SEQ ID NO: 218) OL 2164CAGTACGATAGAAACCCGGC 40 IEX02 PE38 S253N AGATTGCTGCGCGGTACGTA(SEQ ID NO: 219) OL 2165 CAGTACGATAGAAACCCGGC 40 IEX02 PE38 S253KAGCTTGCTGCGCGGTACGTA (SEQ ID NO: 220) OL 2166 CAGTACGATAGAAACCCGGC 40IEX02 PE38 S253P AGAGGGCTGCGCGGTACGTA (SEQ ID NO: 221) OL 2167CAGTACGATAGAAACCCGGC 40 IEX02 PE38 S253T AGGGTGCTGCGCGGTACGTA(SEQ ID NO: 222) OL 2168 GTACGTGCTCGTAGCAGAGAC 33 IEX02 PE38 Q206RCTGGATGCCATC (SEQ ID NO: 223) OL 2169 GATGGCATCCAGGTCTCTGCT 33IEX02 PE38 Q206R ACGAGCACGTAC (SEQ ID NO: 224) OL 2170CGTAGCCAGGACCTGAAGGC 33 IEX02 PE38 D209K CATCTGGCGTGGC (SEQ ID NO: 225)OL 2171 GCCACGCCAGATGGCCTTCAG 33 IEX02 PE38 D209K GTCCTGGCTACG(SEQ ID NO: 226) OL 2183 GAAGCTGCTCAGTCTGCCGTG 32IEX02 PE38 I196A FOR, to TTCGGTGGCGT pair with OL2161 (SEQ ID NO: 227)OL 2184 ACGCCACCGAACACGGCAGA 32 IEX02 PE38 I196A REV, to CTGAGCAGCTTCpair with OL2268 (SEQ ID NO: 228) OL 2185 GAAGCTGCTCAGTCTAACGYG 32IEX02 PE38 I196N FOR, to TTCGGTGGCGT pair with OL2161 (SEQ ID NO: 229)OL 2186 ACGCCACCGAACACGTTAGA 32 IEX02 PE38 I196N REV, to CTGAGCAGCTTCpair with OL2268 (SEQ ID NO: 230) OL 2187 GGTGATGGCGGCGATGCCTCT 33IEX02 to introduce I153A TTTTCTACCCGC FOR (SEQ ID NO: 231) OL 2188GCGGGTAGAAAAAGAGGCAT 33 IEX02 to introduce I153A CGCCGCCATCACC REV(SEQ ID NO: 232) OL 2189 GGTGATGGCGGCGATACCTCT 33IEX02 to introduce I153T TTTTCTACCCGC FOR (SEQ ID NO: 233) OL 2190GCGGGTAGAAAAAGAGGTAT 33 IEX02 to introduce I153T CGCCGCCATCACC REV(SEQ ID NO: 234) OL 2191 GGTGATGGCGGCGATCACTCT 33IEX02 to introduce I153H TTTTCTACCCGC FOR (SEQ ID NO: 235) OL 2192GCGGGTAGAAAAAGAGTGAT 33 IEX02 to introduce I153H CGCCGCCATCACC REV(SEQ ID NO: 236) OL 2193 GCACCCAGAACTGGAGAGTT 32IEX02 to introduce T164R GAACGTCTGCTG FOR (SEQ ID NO: 237) OL 2194CAGCAGACGTTCAACTCTCCA 32 IEX02 to introduce T164R GTTCTGGGTGC REV(SEQ ID NO: 238) OL 2216 CATGGTGGCTCTCCTTCTTAA 39IEX02 Linker to optimize AGTTAAACAAAATTATTT Kozak in pET14b, to anneal(SEQ ID NO: 239) with OL2217 OL 2217 CTAGAAATAATTTTGTTTAAC 39IEX02 Linker to optimize TTTAAGAAGGAGAGCCAC Kozak in pET14b, to anneal(SEQ ID NO: 240) with OL2216 OL 2268 ATCTCCCTCTAGAAATAATTT 38IEX02 outside FOR spanns TGTTTAACTTTAAGAAG over XbaI site (pET14b) - to(SEQ ID NO: 241) be paired with OL2161 OL 2279 GAAGCTGCTCAGTCTATCGTG 32IEX02 FOR oligo to remove TTCGGTGGCGT TM to be paired with(SEQ ID NO: 242) OL2161 OL 2280 ACGCCACCGAACACGATAGA 32IEX02 REV oligo to CTGAGCAGCTTC remove TM to be paired (SEQ ID NO: 243)with OL2268 OL 2281 CTCTGCTACGAGCACGGGCGC 32 IEX02 A201 REV, ONLYCACCGAACACG for templates having Q206 (SEQ ID NO: 244) OL 2282CGTGTTCGGTGGCGCCCGTGC 32 IEX02 A201 FOR, ONLY TCGTAGCAGAGfor templates having Q206 (SEQ ID NO: 245) OL 2283 CATCCAGGTCTCTGCTGGCAG33 IEX02 A204 REV, ONLY CACGTACGCCAC for templates having Q206(SEQ ID NO: 246) OL 2284 GTGGCGTACTGTCTGCCAGCA 33 IEX02 A204 FOR, ONLYGAGACCTGGATG for templates having Q206 (SEQ ID NO: 247) OL 2285CATCCAGGTCTCTGCTCTGAG 33 IEX02 Q204 REV, ONLY CACGTACGCCACfor templates having Q206 (SEQ ID NO: 248) OL 2286 GTGGCGTACGTGCTCAGAGCA33 IEX02 Q204 FOR, ONLY GAGACCTGGATG for templates having Q206(SEQ ID NO: 249) OL 2287 CCAGTTCTGGGTGCCGGCGGT 31 IEX02 A158 REVAGAAAAAGAG (SEQ ID NO: 250) OL 2288 CTCTTTTTCTACCGCCGGCAC 31IEX02 A158 FOR CCAGAACTGG (SEQ ID NO: 251) OL 2289 CCAGTTCTGGGTGCCCTGGGT36 IEX02 Q158 REV AGAAAAAGAGATATC (SEQ ID NO: 252) OL 2290GATATCTCTTTTTCTACCCAG 36 IEX02 Q158 FOR GGCACCCAGAACTGG (SEQ ID NO: 253)OL 2291 GTCCAGTTCTGGGTGGAGCGG 38 IEX02 S159 REV GTAGAAAAAGAGATATC(SEQ ID NO: 254) OL 2292 GATATCTCTTTTTCTACCCGCT 38 IEX02 S159 FORCCACCCAGAACTGGAC (SEQ ID NO: 255) OL 2293 ACCACCCAGAACTGGACCGTT 25IEX02 T159 REV GAAC (SEQ ID NO: 256) OL 2294 CCAGTTCTGGGTGGTGCGGGT 31IEX02 T159 FOR AGAAAAAGAG (SEQ ID NO: 257) OL 2295 GAGCTTGGGTCCAGATCGCCA23 IEX02 generic REV oligo CC for mutations at 333 (SEQ ID NO: 258) OL2296 CTGGACCCAAGCTCTGCCCCG 31 IEX02 A333 FOR GATAAAGAAC (SEQ ID NO: 259)OL 2297 CTGGACCCAAGCTCTAACCCG 28 IEX02 N333 FOR GATAAAG (SEQ ID NO: 260)OL 2298 CTGGACCCAAGCTCTACCCCG 28 IEX02 T333 FOR GATAAAG (SEQ ID NO: 261)OL 2299 CTGGACCCAAGCTCTCAGCCG 31 IEX02 Q333 FOR GATAAAGAAC(SEQ ID NO: 262) OL 2300 CTGGACCCAAGCTCTCACCCG 28 IEX02 H333 FOR GATAAAG(SEQ ID NO: 263) OL 2301 CTGGACCCAAGCTCTATCCCG 48 IEX02 N338 FORGATAAAGAAAACGCTATTTCT GCCCTG (SEQ ID NO: 264) OL 2302CTGGACCCAAGCTCTATCCCG 46 IEX02 E338 FOR GATAAAGAAGAGGCTATTTCT GCCC(SEQ ID NO: 265) OL 2303 CTGGCTACGAGCACGGGCGC 28 IEX02 V201A REVCACCGAAC (SEQ ID NO: 266) OL 2304 GTTCGGTGGCGCCCGTGCTCG 28IEX02 V201A FOR TAGCCAG (SEQ ID NO: 267) OL 2305 CTTCAGGTCCTGGCTGGCAGC30 IEX02 R204A REV, ONLY ACGTACGCC for templates having D209K(SEQ ID NO: 268) OL 2306 GGCGTACGTGCTGCCAGCCAG 30 IEX02 R204A FOR, ONLYGACCTGAAG for templates having D209K (SEQ ID NO: 269) OL 2307CTTCAGGTCCTGGCTCTGAGC 29 IEX02 R204Q REV, ONLY ACGTACGCfor templates having D209K (SEQ ID NO: 270) OL 2308 GCGTACGTGCTCAGAGCCAG29 1EX02 R204Q FOR, ONLY GACCTGAAG for templates having D209K(SEQ ID NO: 271) OL 2309 GTTCAACGGTCCAGTTGTTGG 33 IEX02 Q161N REVTGCCGCGGGTAG (SEQ ID NO: 272) OL 2310 CTACCCGCGGCACCAACAACT 33IEX02 Q161N FOR GGACCGTTGAAC (SEQ ID NO: 273) OL 2311GTTCAACGGTCCAGTTGGTGG 33 IEX02 Q161T REV TGCCGCGGGTAG (SEQ ID NO: 274)OL 2312 CTACCCGCGGCACCACCAACT 33 IEX02 Q161T FOR GGACCGTTGAAC(SEQ ID NO: 275) OL 2313 CAGCAGACGTTCAACGGCCC 31 IEX02 T164A REVAGTTCTGGGTG (SEQ ID NO: 276) OL 2314 CACCCAGAACTGGGCCGTTGA 31IEX02 T164A FOR ACGTCTGCTG (SEQ ID NO: 277) OL 2315GACGTTCAACGGTCCAGGCCT 33 IEX02 N162A REV GGGTGCCGCGGG (SEQ ID NO: 278)OL 2316 CCCGCGGCACCCAGGCCTGG 33 IEX02 N162A FOR ACCGTTGAACGTC(SEQ ID NO: 279) OL 2318 ATTGCCACCATGGCGGAAGTG 22 IEX02 FOR RFB4 (NcoI)C  (SEQ ID NO: 280) OL 2320 CACCAGGCCGCTGCTTTTGAT 31IEX02 REV to create RBF4 CTCCAGCTTG for RFB4-PE38-8xHis to(SEQ ID NO: 281) pair with OL2318 OL 2321 CAAGCTGGAGATCAAAAGCA 31IEX02 FOR to create GCGGCCTGGTG RFB4-PE38-8xHis to pair (SEQ ID NO: 282)with OL2322 OL 2322 CGATTCTCGAGTTACTTCAGG 66 IEX02 REV introducingTCCTCGTGGTGGTGGTGATGA 8xHis C-terminus of PE, TGATGATGACGCGGCGGTTTAintroducing XhoI CCC (SEQ ID NO: 283) OL 2323 CAAGCTGGAGATCAAAGCTC 44IEX02 FOR to create ATGGGGGCAGCCATCATCATC RFB4-6xHis PE38 fusions ATC(pIEX02-302 and pIEX02- (SEQ ID NO: 284) 304) to pair with OL2161 OL2324 GATGATGATGATGGCTGCCCC 44 IEX02 REV to create CATGAGCTTTGATCTCCAGCTRFB4-6xHis PE38 fusions TG  (pIEX02-302 and pIEX02- (SEQ ID NO: 285)304) to pair with OL2318

Example 10: Analysis of Genes Encoding Amino Acid Substituted Forms ofPE by an In Vitro Transcription/Translation (IVTT) Assay

The cell-free in vitro transcription/translation (IVTT) assay wasperformed with a TnT® T7 Coupled Reticulocyte Lysate System (PROMEGA®catalog # L4610) following the procedure described in the User's Manualprovided in the kit. See, PROMEGA®, Technical Bulletin #TB126, Revised12/10, pp. 1-28 (2010) which is incorporated by reference herein.

WT PE in plasmid pET14b-K was used as standard on every plate and testedat concentrations ranging from 0.08 ng to 10 ng in a 12.5 microliterfinal volume reaction. All test samples were run in triplicate. DNAsencoding WT or amino acid substituted PE in plasmid pET14b-K were addedto the IVTT reaction mix supplemented with NAD+ (final concentration0.15 mM; Fisher Scientific catalog # BPE9746-212) and incubated at 30°C. for 15 min. Following a subsequent cooling step at 4° C. for 5 min,250 ng of T7 Luciferase plasmid (Luciferase T7 control DNA supplied inthe TnT® T7 Coupled Reticulocyte Lysate System) was added to eachreaction and incubated at 30° C. for 90 min. The reaction was stopped byplacing the samples on ice. Samples were analyzed using the STEADY-GLO®Luciferase Assay (PROMEGA® catalog # E2510) according to the protocolprovided by the manufacturer. See, PROMEGA®, Technical Bulletin #TM051,revised 9/11, pp. 1-23 (2011) which is incorporated by reference herein.Luminescence was measured in a FLUOstar OPTIMA plate reader (BMG LabtechLtd., Aylesbury, UK)

A representative result is shown in FIG. 10 which shows the resultsexpressed in CPS (counts per second as read from the FLUOstar OPTIMAfluorescence plate reader) for luciferase activity from IVTT assays ofgenes encoding either WT PE (pIEX02-001 (SEQ ID NO: 1)) or encodingamino acid substituted PE (pIEX02-228 (SEQ ID NO: 177), pIEX02-244 (SEQID NO:178), pIEX02-246 (SEQ ID NO:179)); which were expressed as fusionproteins comprising a histidine polymer and a linker sequence precedinga sequence of WT PE or amino acid substituted PE; see, pIEX02-001 PE WT(SEQ ID NO:189 (DNA) and SEQ ID NO:190 (AA)), pIEX02-228 amino acidsubstituted PE (SEQ ID NO:193 (DNA) and SEQ ID NO:194 (AA)), pIEX02-244amino acid substituted PE (SEQ ID NO:197 (DNA) and SEQ ID NO:198 (AA)),pIEX02-246 amino acid substituted PE (SEQ ID NO:201 (DNA) and SEQ IDNO:202 (AA)). See also, Table 13.

For the analysis of various amino acid substituted PEs, the potency ofeach mutated PE in inhibiting IVTT was expressed as relative inhibitionexhibited via expression from 2.5 nanograms of DNA encoding amino acidsubstituted PE compared to expression from 2.5 nanograms WT PE DNA asshown in Table 13 (data expressed as % inhibition of IVTT for the DNAencoding amino acid substituted PE compared to wild-type PE). In orderto identify “inhibitory” amino acid substituted PE polypeptides (i.e.,genes encoding amino acid substituted forms of PE which inhibit IVTT),selected mutations in each T cell epitope as identified in Table 11 wereinitially tested using various combinations of epitope 5 mutations(e.g., corresponding to S241N, S241K, S241P and S241T in SEQ ID NO:1)along with mutations in either: epitope 4; epitopes 4 and 3; epitopes 4and 1; epitopes 4 and 2; or, epitopes 4 and 6 (see, Table 13). For allcombinations except those including amino acid substitutions in epitope3 at 1184 (SEQ ID NO:1; or, 1196 in SEQ ID NO:2) (which produced 0%inhibition), one or more inhibitory PE polypeptides were identified(Table 13). (Note: “Inhibitory PE polypeptides” indicates amino acidsubstituted forms of PE which exhibit PE biological activity in theinhibition of IVTT). From structural analysis of PE, it was noted thatresidue 1184 (SEQ ID NO:1; or, 1196 in SEQ ID NO:2) (anchor residue 1,Table 11) was located within the active enzymatic site of PE. In view ofthis result, alternative mutations distal to the active site were soughtat anchor residues 6 and 9 (V189 and R192 in SEQ ID NO:1; or, V201 andR204 in SEQ ID NO:2). Alternative epitope 3 mutations at V189 and R192in SEQ ID NO:1 were tested in combination with other epitope mutations.These mutations confirmed that inhibitory PE polypeptides with epitope 3mutations could also be generated (Table 13, pIEX02-173 to -248). Arange of combinations of DNAs encoding multiple amino acid substitutedforms of PE were tested progressively leading to a final analysis ofmutations in four of six, five of six, and six of six identifiedimmunogenic epitopes. See, Table 13, “Quadruplicates”, “Quintuplicates”and “Sextuplicates”. In this regard, quadruplicate epitope mutationswere identified which exhibited IVTT inhibitory activity ranging from 0%to about 70%. Quintuplicate mutations were identified that exhibitedIVTT inhibitory activity ranging from about 5% to about 35%.Sextuplicate mutations were identified that exhibited IVTT inhibitoryactivity ranging from about 5% to about 20%. It is also noted thatmultiple single, double, and triple epitope mutations also resulted inamino acid substituted forms of PE exhibiting PE biological activity inthe inhibition of IVTT such that the percent (%) inhibitory activityranged from 0% to 100% (or about 100%); see Table 13.

Three different “candidates” (i.e., amino acid substituted forms of PEor DNA constructs encoding the same) were selected for use as examplesin performing subsequent experiments described further herein. Inparticular, additional experiments were performed using the sextuplicateAA substituted candidate pIEX02-244 (SEQ ID NO: 178; see also, Table13); which retained approximately 20% of the WT PE inhibitory activity.Likewise, additional experiments were also performed using thesextuplicate AA substituted candidate pIEX02-246 (SEQ ID NO: 179; seealso, Table 13) which retained approximately 8% of the WT PE inhibitoryactivity; and using the quintuplicate AA substituted candidatepIEX02-228 (SEQ ID NO: 177; see also, Table 13) which retainedapproximately 36% of the WT PE inhibitory activity. These were expressedas fusion proteins comprising a histidine polymer and a linker sequencepreceding a sequence of WT PE or amino acid substituted PE; see,pIEX02-001 PE WT (SEQ ID NO:189 (DNA) and SEQ ID NO:190 (AA)),pIEX02-228 amino acid substituted PE (SEQ ID NO:193 (DNA) and SEQ IDNO:194 (AA)), pIEX02-244 amino acid substituted PE (SEQ ID NO:197 (DNA)and SEQ ID NO:198 (AA)), pIEX02-246 amino acid substituted PE (SEQ IDNO:201 (DNA) and SEQ ID NO:202 (AA)). These AA substituted PEpolypeptides, and DNA constructs encoding, them may be referenced hereinas “228”, “244” or “246” using simply these three numbers, or usingthese numbers and a prefix or suffix included therewith.

Moreover, it is noted that in view of the highly cytotoxic nature ofwild-type PE, IVTT inhibition activity (i.e., cytotoxicity) as low asabout 5% (or higher) of WT (e.g., 8%, 20%, and 36%) in amino acidsubstituted forms of PE may provide a therapeutically effectivepolypeptide. See, for example, Thomas et al., “Abrogation of Head andNeck Squamous Cell Carcinoma Growth by Epidermal Growth Factor ReceptorLigand Fused to Pseudomonas Exotoxin Transforming Growth Factor α-PE38,”Clin. Cancer Res. 10:7079-7087 (2004); Siegall et al., “Cell-specifictoxicity of a chimeric protein composed of interleukin-6 and Pseudomonasexotoxin (IL6-PE40) on tumor cells”, Mol. Cell. Biol. 10(6); 2443-2447(1990); and, Weldon & Pastan, “A Guide to Taming a Toxin—RecombinantImmunotoxins Constructed From Pseudomonas Exotoxin A for the Treatmentof Cancer”, FEBS Journal 278(23):4683-4700 (2011).

TABLE 13 Examples of Amino Acid Substituted Forms of PE and AssociatedCell Cytotoxic Activity. Epitopes pIEX02 - Epitope Epitope EpitopeEpitope Epitope Epitope % Inhibition changed ### 5 4 3 1 2 6 of IVTTWild-Type (WT) 001 100.00 (WT) Single Substitutions 5 003 S241N 100.00[S253N] 5 004 S241K 100.00 [S253K] 5 005 S241P 99.71 [S253P] 5 006 S241T99.99 [S253T] Double Substitutions 4, 5 007 S241N Q194R 41.63 [S253N][Q206R] 4, 5 008 S241K Q194R 69.51 [S253K] [Q206R] 4, 5 009 S241P Q194R20.58 [S253P] [Q206R] 4, 5 010 S241T Q194R 21.44 [S253T] [Q206R] 4, 5011 S241N D197K 99.88 [S253N] [D209K] 4, 5 012 S241K D197K 42.49 [S253K][D209K] 4, 5 013 S241P D197K 74.87 [S253P] [D209K] 4, 5 014 S241T D197K98.84 [S253T] [D209K] Triple Substitutions 3, 4, 5 015 S241N Q194R I184A0.00 [S253N] [Q206R] [I196A] 3, 4, 5 016 S241K Q194R I184A 0.00 [S253K][Q206R] [I196A] 3, 4, 5 017 S241P Q194R I184A 0.00 [S253P] [Q206R][I196A] 3, 4, 5 018 S241T Q194R I184A 0.00 [S253T] [Q206R] [I196A] 3, 4,5 019 S241N Q194R I184N 0.00 [S253N] [Q206R] [I196N] 3, 4, 5 020 S241KQ194R I184N 0.00 [S253K] [Q206R] [I196N] 3, 4, 5 021 S241P Q194R I184N0.00 [S253P] [Q206R] [I196N] 3, 4, 5 022 S241T Q194R I184N 0.00 [S253T][Q206R] [I196N] 3, 4, 5 024 S241K D197K I184A 0.00 [S253K] [D209K][I196A] 3, 4, 5 027 S241N D197K I184N 0.00 [S253N] [D209K] [I196N] 3, 4,5 028 S241K D197K I184N 0.00 [S253K] [D209K] [I196N] 3, 4, 5 029 S241PD197K I184N 0.00 [S253P] [D209K] [I196N] 3, 4, 5 030 S241T D197K I184N0.00 [S253T] [D209K] [I196N] 1, 4, 5 127 S241T Q194R I141A 15.58 [S253T][Q206R] [I153A] 1, 4, 5 128 S241T Q194R I141T 14.16 [S253T] [Q206R][I153T] 1, 4, 5 129 S241T Q194R I141H 56.73 [S253T] [Q206R] [I153H] 1,4, 5 130 S241T D197K I141H 20.46 [S253T] [D209K] [I153H] 1, 4, 5 131S241T D197K I141T 86.84 [S253T] [D209K] [I153T] 1, 4, 5 132 S241T D197KI141A 88.15 [S253T] [D209K] [I153A] 1, 4, 5 139 S241T D197K R146Q 21.81[S253T] [D209K] [R158Q] 1, 4, 5 140 S241T D197K G147S 42.81 [S253T][D209K] [G159S] 1, 4, 5 143 S241T D197K Q149T 49.33 [S253T] [D209K][Q161T] 1, 4, 5 170 S241T Q194R R146A 39.74 [S253T] [Q206R] [R158A] 1,4, 5 171 S241T Q194R R146Q 6.01 [S253T] [Q206R] [R158Q] 1, 4, 5 172S241T Q194R G147S 1.49 [S253T] [Q206R] [G159S] 2, 4, 5 144 S241T D197KT152A 100.67 [S253T] [D209K] [T164A] 2, 4, 5 145 S241T D197K N150A 70.69[S253T] [D209K] [N162A] 2, 4, 5 133 S241T Q194R T152R 17.46 [S253T][Q206R] [T164R] 2, 4, 5 134 S241T D107K T152R 23.78 [S253T] [D209K][T164R] 6, 4, 5 146 S241T D197K I321A 104.47 [S253T] [D209K] [I333A] 6,4, 5 147 S241T D197K I321N 99.97 [S253T] [D209K] [I333N] 6, 4, 5 148S241T D197K I321T 53.19 [S253T] [D209K] [I333T] 6, 4, 5 149 S241T D197KI321Q 99.91 [S253T] [D209K] [I333Q] 6, 4, 5 150 S241T D197K I321H 89.43[S253T] [D209K [I333H] 6, 4, 5 152 S241T D197K Q326E 99.97 [S253T][D209K] [Q338E] 3, 4, 5 173 S241T Q194R R192A 23.15 [S253T] [Q206R][R204A] 3, 4, 5 174 S241T Q194R R192Q 16.37 [S253T] [Q206R] [R204Q] 3,4, 5 175 S241T Q194R V189A 6.50 [S253T] [Q206R] [V201A] QuadruplicateSubstitutions 1, 2, 4, 5 156 S241N Q194R I141T T152R 6.16 [S253N][Q206R] [I153T] [T164R] 1, 2, 4, 5 166 S241N D197K I141A T152R 7.90[S253N] [D209K] [I153A] [T164R] 1, 3, 4, 5 105 S241N D197K I184A I141A0.00 [S253N] [D209K] [I196A] [I153A] 1, 3, 4, 5 106 S241K D197K I184AI141A 0.00 [S253K] [D209K] [I196A] [I153A] 1, 3, 4, 5 110 S241K D197KI184A I141T 0.00 [S253K] [D209K] [I196A] [I153T] 1, 3, 4, 5 111 S241PD197K I184A I141T 0.00 [S253P] [D209K] [I196A] [I153T] 1, 3, 4, 5 112S241T D197K I184A I141T 0.00 [S253T] [D209K] [I196A) [I153T] 1, 3, 4, 5114 S241K D197K I184A I141H 0.00 [S253K] [D209K] [I196A] [I153H] 1, 3,4, 5 115 S241P D197K I184A I141H 0.00 [S253P] [D209K] [I196A] [I153H] 1,3, 4, 5 117 S241N D197K I184N I141A 0.00 [S253N] [D209K] [I196N] [I153A]1, 3, 4, 5 118 S241K D197K I184N I141A 0.00 [S253K] [D209K] [I196N][I153A] 1, 3, 4, 5 120 S241T D197K I184N I141A 0.00 [S253T] [D209K][I196N] [I153A] 1, 3, 4, 5 121 S241N D197K I184N I141T 0.00 [S253N][D209K] [I196N] [I153T] 1, 3, 4, 5 122 S241K D197K I184N I141T 0.00[S253K] [D209K] [I196N] [I153T] 1, 3, 4, 5 123 S241P D197K I184N I141T0.00 [S253P] [D209K] [I196N] [I153T] 1, 3, 4, 5 124 S241T D197K I184NI141T 0.00 [S253T] [D209K] [I196N] [I153T] 1, 3, 4, 5 125 S241N D197KI184N I141H 0.00 [S253N] [D209K] [I196N] [I153H] 2, 3, 4, 5 179 S241TQ194R V189A T152R 13.37 [S253T] [Q206R] [V201A] [T164R] 2, 3, 4, 5 180S241T Q194R R192A T152R 58.93 [S253T] [Q206R] [R204A] [T164R] 2, 3, 4, 5181 S241T Q194R R192Q T152R 13.70 [S253T] [Q206R] [R204Q] [T164R] 1, 3,4, 5 183 S241T D197K R192A I141A 36.87 [S253T] [D209K] [R204A] [I153A]1, 3, 4, 5 188 S241T D197K R192A I141T 20.75 [S253T] [D209K] [R204A][I153T] 1, 2, 4, 5 195 S241T D197K I141T T152A 42.90 [S253T] [D209K][I153T] [T164A] 1, 4, 5, 6 200 S241T D197K I141T I321A 22.04 [S253T][D209K] [I153T] [I333A] 1, 4, 5, 6 201 S241T D197K I141T I321N 58.30[S253T] [D209K] [I153T] [I333N] 1, 4, 5, 6 204 S241T D197K I141T I321H12.76 [S253T [D209K] [I153T] [I333H] 1, 2, 4, 5 208 S241T D197K I141AT152A 49.49 [S253T] [D209K] [I153A] [T164A] 1, 4, 5, 6 213 S241T D197KI141A I321A 18.03 [S253T] [D209K] [I153A] [I333A] 1, 4, 5, 6 214 S241TD197K I141A I321N 0.18 [S253T] [D209K] [I153A] [I333N] 1, 4, 5, 6 215S241T D197K I141A I321T 5.87 [S253T] [D209K] [I153A] [I333T] 1, 4, 5, 6216 S241T D197K I141A I321Q 20.21 [S253T] [D209K] [I153A] [I333Q] 1, 4,5, 6 217 S241T D197K I141A I321H 11.22 [S253T] [D209K] [I153A] [I333H]1, 4, 5, 6 219 S241T D197K I141A Q326E 70.65 [S253T] [D209K] [I153A][Q338E] Quintuplicate Substitutions 222 S241T D197K G147S T152A Q326E4.87 [S253T] [D209K] [G159S] [T164A] [Q338E] 224 S241T D197K Q149T T152AQ326E 3.69 [S253T] [D209K] [Q161T] [T164A] [Q338E] 226 S241T D197K I141AN150A Q326E 11.23 [S253T] [D209K] [I153A] [N162A] [Q338E] 1, 2, 4, 5, 6228 S241T D197K I141A T152R Q326E 36.27 [S253T] [D209K] [I153A] [T164R][Q338E] 1, 2, 4, 5, 6 229 S241T D197K I141A T152A Q326E 18.11 [S253T][D209K] [I153A] [T164A] [Q338E] 1, 2, 3, 4, 5 221 S241T Q194R R192AI141A T152R 4.79 [S253T] [Q206R] [R204A] [I153A] [T164R] 1, 2, 4, 5, 6242 S241T D197K I141T T152A Q326E 21.64 [S253T] [D209K] [I153T] [T164A][Q338E] Sextuplicate Substitutions 1-6 244 S241T D197K R192A I141T T152AQ326E 20.53 [S253T] [D209K] [R204A] [I153T] [T164A] [Q338E] 1-6 246S241T D197K R192A I141A T152A Q326E 7.95 [S253T] [D209K] [R204A] [I153A][T164A] [Q338E] 1-6 248 S241T D197K R192A I141A T152R Q326E 4.45 [S253T][D209K] [R204A] [I153A] [T164R] [Q338E] Note: Non-bracketed amino acidsubstitution positions correspond to the PE polypeptide sequence in SEQID NO: 1. Bracketed [amino acid substitution positions] correspond tothe PE polypeptide sequence in SEQ ID NO: 2 (comprising an N-terminal12-amino acid linker).

Example 11: Ex Vivo Human T Cell Assay to Assess Immunogenicity ofWild-Type (WT) and Amino Acid Substituted PE

Ex vivo human T cell assays (EPISCREEN™; see e.g., preceding Examples)were performed to assess the immunogenicity of whole proteinscorresponding to pIEX02-244 (SEQ ID NO:178) pIEX02-246 (SEQ ID NO:179)and pIEX02-228 (SEQ ID NO:177) (Example 10). In order to avoid directcytotoxicity to cells used in the assay, “null mutants” were generatedfor the three candidates and WT PE by overlapping PCR as in Example 9using primers OL2162 and 2163 (Table 12) to introduce an amino acidsubstitution of E287D in the three candidates and WT PE (to give SEQ IDNOs: 180 to 183). (Note: “Null mutants” is intended to indicate mutatedforms of PE which lack cell cytotoxic biological activity; the aminoacid substitution used to generate null mutants corresponds to a changeof E287D in SEQ ID NO:180; or E299 in SEQ ID NO:2.) The E287D (SEQ IDNOs: 180 to 183) encoding genes were cloned into pET14b-K as in Example9. WT PE sequence is shown in SEQ ID NO:180; pIEX02-228 sequence isshown in SEQ ID NO:181; pIEX02-244 sequence is shown in SEQ ID NO:182;and, pIEX02-246 sequence is shown in SEQ ID NO:183. These were expressedas fusion proteins comprising a histidine polymer and a linker sequencepreceding a “null mutant” sequence of WT PE or amino acid substitutedPE; see, pIEX02-001 PE WT null mutant (SEQ ID NO: 191 (DNA) and SEQ IDNO:192 (AA)), pIEX02-228 null mutant (SEQ ID NO:195 (DNA) and SEQ ID NO:196 (AA)), pIEX02-244 null mutant (SEQ ID NO: 199 (DNA) and SEQ IDNO:200 (AA)), pIEX02-246 null mutant (SEQ ID NO:203 (DNA) and SEQ IDNO:204 (AA)).

The host cell for expression of the PE E287D genes was an Escherichiacoli BL21 derivative strain called SHuffle™ T7 Express (NEB catalog#C3029H, New England Biolabs UK Ltd., Knowl Piece, UK) which was alteredto overexpress the chaperonins GroEL/ES by amplification of the GroEL/ESoperon, including its promoter/regulatory sites, from E. coli DH5alpha™(Invitrogen catalog #18265-017, Life Technologies Ltd., Paisley, UK)using OL2097 (introducing EagI site) and OL2098, introducing HindIIIsite (Table 12). The resulting PCR fragment was subcloned into pACYC184(NEB catalog #E4152S) which was then transformed into SHuffle™ T7 withselection for chloramphenicol resistance. The PE E287D (SEQ ID NOs: 180to 183) genes in pET14b-K were transformed into the SHuffle™ T7/GroEL/ESstrain with selection for ampicillin resistance. Single colonies weregrown in 2×YT medium (Sigma-Aldrich catalog # Y2627-1KG) and proteinexpression was induced at OD_(600nm) 1.0 by addingisopropyl-3-D-thio-galactoside (IPTG) to a final concentration of 0.4mM. Cultures were then grown at 16 degrees C. for 17 h before harvestingby centrifugation. Cell pellets were resuspended in 35 ml of bindingbuffer (50 mM Tris pH 8.0, 500 mM NaCl and 10 mM imidazole) supplementedwith protease inhibitors (cOmplete protease inhibitor tablets, Rochecatalog #11873580001, Roche Diagnostics Ltd., Burgess Hill, UK (mixtureof several protease inhibitors for inhibition of serine and cysteineproteases)). Cells were lysed by sonication (SONICATOR®, Misonix catalog# XL2020, Misonix Inc., Farmingdale, N.Y.), and cell debris andinsoluble material removed by centrifugation. Proteins were purifiedfrom the soluble fraction by nickel chelate affinity chromatographyusing HISTRAP® FF Crude columns (GE Healthcare catalog #11-004-58, GEHealthcare Life Sciences, Little Chalfont, UK). After loading, thecolumns were washed with 50 mM Tris (pH 8.0) containing 500 mM NaCl and20 mM imidazole and bound protein was eluted with 50 mM Tris (pH 8.0)containing 500 mM NaCl and 500 mM imidazole. Following buffer exchangeto 20 mM Tris (pH 8.0) using Zeba Spin desalting columns (7K MWCO,Pierce catalog #89893), a negative purification step was employed usinganion exchange chromatography on Q-Sepharose (1 ml, HISTRAP® Q FF column(GE Healthcare catalog #17-5053-01) with 20 mM Tris pH 8.0 and an NaClgradient form 0 M to 1.5 M. For each protein, the column flow throughwas concentrated using an AMICON® Ultra centrifugal filter (EMDMillipore catalog # UFC 800 396, EMD Millipore, Feltham, UK) and furtherpurified by size-exclusion chromatography (120 ml, HiLoad 16/60SUPERDEX® 75 pg (GE Healthcare catalog #28-9893-33)) using 1×PBS pH 7.4(PAA catalog # H15-002, PAA Laboratories Ltd, Yeovil, UK). For eachprotein, the protein peak was collected and concentrated.

Endotoxin levels were determined using an ENDOSAFE®-PTS™ Portable TestSystem reader (Charles River Laboratories Inc., Wilmington, Mass.) withENDOSAFE® Licensed PTS Endotoxin cartridges (Charles River catalog #PTS20F). Endotoxins were reduced to a value below 5 endotoxin units(EU)/mg by repeatedly performing a phase separation using TritonX-114,(Aida Y. and Pabst M. J., Journal of Immunological Methods, 132 (1990)191-195). Triton X-114 was removed using PIERCE® Detergent Removal SpinColumns according to the manufacturer's provided protocol (PIERCE®catalog #87779; Thermo Fisher Scientific/PIERCE® Biotechnology,Rockford, Ill.; see, Thermo Scientific Instructions manual #2164.3).Protein concentration was quantified by absorbance at 280 nm using aBIOMATE™ 3 UV-Visible spectrophotometer (Thermo Fisher Scientific) and aconversion factor of OD₂₈₀ 1.0=1.15 mg/ml derived from the calculatedmolar extinction coefficient of 6×His PE (Pace C. N. et al. ProteinScience 1995 4:2411-2423).

E vivo human T cell assays (EPISCREEN®) were performed using PBMCisolated from healthy community donor buffy coats as in Example 2. Acohort of 20 donors was selected to best represent the number andfrequency of HLA-DR allotypes expressed in the world population. Thehaplotypes of the 20 donors in the assay is shown in Table 14. PBMCsfrom each donor were thawed, counted and viability assessed. Cells wererevived in room temperature AIM-V® culture medium (INVITROGEN®, Paisley,UK), washed and resuspended in AIM-V® to 4-6×10⁶ PBMC/ml. For eachdonor, 1 ml of cells were dispensed into multiple wells of a 24 wellplate. 0.5 ml of proteins were added at 50 micrograms/ml per sampletogether with 0.5 ml of AIM-V® culture medium. For each donor, areproducibility control (cells incubated with 100 micrograms/ml keyholelimpet hemocyanin (KLH), an “intermediate” positive control (expected togive 20-30% T cell responses) of humanized A33 antibody (Welt et al.Clinical Cancer Research, 9 (2003) p 1338-1343) (cells were incubatedwith 50 micrograms/ml humanized A33), and a culture medium only controlwell were also included. Cultures were incubated for a total of 8 daysat 37° C. with 5% CO₂.

TABLE 14 Donor Haplotypes Donor KLH No Haplotype Test 1 IEX02 1 DRB1*01,DRB1*11; DRB3* 1.95 5.41 2 DRB1*11, DRB1*15; DRB3*; DRB5* N/D 8.39 3DRB1*04, DRB1*11; DRB3*; DRB4* 6.04 4.58 4 DRB1*08, DRB1*14; DRB3* 1.781.35 5 DRB1*07, DRB1*13; DRB3*; DRB4* 5.57 6.77 6 DRB1*04; DRB4* 12.3611.25 7 DRB1*03, DRB1*04; DRB3*; DRB4* 1.48 1.12 8 DRB1*03, DRB1*13;DRB3* 2.73 1.63 9 DRB1*03, DRB1*07; DRB3*; DRB4* 3.59 3.07 10 DRB1*04,DRB1*12; DRB3*; DRB4* 3.35 3.26 11 DRB1*01, DRB1*07 13.67 15.34 12DRB1*01, DRB1*14; DRB3* 6.05 50.13 13 DRB1*07, DRB1*09; DRB4* 9.17 19.3214 DRB1*15; DRB5* 2.83 3.97 15 DRB1*03, DRB1*15; DRB3*; DRB5* 3.36 3.0916 DRB1*07, DRB1*13; DRB3*; DRB4* 2.18 6.76 17 DRB1*15, DRB1*13, DRB3*;DRB5* 1.93 7.04 18 DRB1*01, DRB1*04; DRB4* 2.49 28.59 19 DRB1*01,DRB1*11; DRB3* 0.83 4.50 20 DRB1*01 2.03 3.18

For the T cell proliferation assay, on days 5, 6, 7 and 8, the cells ineach well were gently resuspended and triplicate 100 microliter aliquotswere transferred to each well of a round bottomed 96 well plate. Thecultures were pulsed with 0.75 microCi [3H]-Thymidine (PERKIN ELMER®,Beaconsfield, UK) in 100 microliters AIM-V® culture medium and incubatedfor a further 18 hours before harvesting onto filter mats (PerkinElmer®) using a TOMTEC® HARVESTER 96™ Mach III cell harvester (TOMTEC®Inc., Hamden, Conn., USA). Counts per minute (cpm) for each well weredetermined using MELTILEX® solid scintillator (PERKIN ELMER® Life andAnalytical Sciences, Shelton, Conn., USA) via scintillation counting ona Wallac 1450 Microbeta Trilux Microplate Scintillation and LuminescenceCounter (Perkin Elmer®) in paralux, low background counting.

For proliferation assays, an empirical Stimulation Index (S) thresholdof equal to, or greater than, 2 (SI≧2.0) was used whereby samplesinducing proliferative responses above this threshold at any day afteraddition of protein were deemed positive. (The Stimulation Index is aratio of stimulated proliferative response compared to a backgroundindex; an SI of I=background or “noise”.) For the triplicateproliferation data for each time point with each protein, thesignificance (p<0.05) of positive responses was defined by statisticaland empirical thresholds by comparing CPM of test protein wells againstmedium-only control wells using unpaired two sample Student's T-Test.

The results of the proliferation assay are shown in Table 15. Theresults demonstrate a significantly reduced level of T cell responsesfrom the amino acid substituted PE molecules: pIEX02-228 (SEQ ID NO:181) 5% donor responses; pIEX02-244 (SEQ ID NO: 182) 10% donorresponses; and, pIEX02-246 (SEQ ID NO:183) 200 donor responses, comparedto WT PE (SEQ ID NO: 180) which induced T cell responses in 70% ofdonors.

TABLE 15 Relative T-cell Stimullated Proliferative Responses to AminoAcid Substituted variants of PE compared to Wild-Type (WT) PE. WTpIEX02- pIEX02- pIEX02- Hu PE 228 244 246 A33 Donor 1 P Donor 2  P* PDonor 3  P* P P P Donor 4 Donor 5 Donor 6 P Donor 7 Donor 8 P Donor 9 PDonor 10 P Donor 11 P P P Donor 12 P P Donor 13 P Donor 14 P P P Donor15 P Donor 16 P P Donor 17 P Donor 18 Donor 19 P P P Donor 20 % Donor 705 10 20 30 Proliferation *Positive T cell responses for proliferation(SI ≧ 2.00, significant p < 0.05) during the entire time course days 5-8(“P”) are shown. **Borderline responses (significant p < 0.05 with SI ≧1.90) are shown (*).

In addition to the proliferation assay, additional analysis of thecytokines IL-2 and IL-6 was performed using aliquots of culturesupernatant taken on day 6. The analysis was performed using the BDCytometric Bead Array (CBA) Enhanced Sensitivity Flex Set Systems forIL-2 and IL-6 (BD Bioscience, Oxford, UK) according to themanufacturer's instructions. The enhanced sensitivity standards from theCBA kit were reconstituted and serially diluted before adding 50microliters of supernatant or standard to 20 microliters of mixedcapture beads in 96 well filter plates (Millipore, Watford, UK) andincubating for 2 hours. Mixed human detection reagent (20 microliters)was then added to each well and incubated for a further 2 hours. Plateswere washed twice and enhanced detection (100 microliters) reagent addedto each well for a final 1 hour incubation. Plates were washed beforereading on an Accuri C6 instrument (BD Biosciences).

Data was analysed using FCAP v3.0 software (BD Biosciences). For eachindividual donor, data was expressed as pg/ml of cytokine for each donorand plotted on a log scale with a median of cytokine levels depicted asa line. The results are shown in FIG. 11 which shows a significantlyreduced level of the cytokines IL-2 and IL-6 from the amino acidsubstituted PE molecules pIEX02-228 (SEQ ID NO:181), pIEX02-244 (SEQ IDNO:182) and pIEX02-246 (SEQ ID NO: 183) compared to WT PE (SEQ ID NO:180).

The proliferation and cytokine results both independently demonstratethat the amino acid substitutions in PE result in greatly reduced levelof T cell responses when using amino acid substituted forms of PE. Theseresults considered and expected to correlate with low or reduced PEimmunogenicity in human subjects.

Example 12: Cytotoxicity Analysis of Amino Acid Substituted PE inDendritic Cells

Amino acid substituted forms of PE and WT PE may be generated as inExample 11. For a dendritic cell cytotoxicity assay, PBMC are isolatedfrom healthy community donor buffy coats (preferably from blood drawnwithin 24 hours), for example, by Lymphoprep (Axis-shield, Dundee, UK)density centrifugation. To prepare monocyte-derived dendritic cells(DC), CD14+ cells (monocytes) may be isolated from donor PBMCpreparations using Miltenyi Monocyte Isolation Kit II (human) and LScolumns (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany; catalog#130-091-153). Monocytes are resuspended in an appropriate culturemedium, such as AIM-V® cell culture medium supplemented with 1000 IU/mlIL-4 and 1000 IU/ml GM-CSF (“DC culture medium”) to 4-6×10⁶ cells/ml andthen distributed in 24 well plates (e.g., 2 ml final culture volume).Cells are fed on day 2 by half volume DC culture medium change. On day3, amino acid substituted PE and WT PE proteins are added to semi-matureDC to selected final concentrations, such as 1 micrograms/ml or 10micrograms/mi. Semi-mature DC are incubated for a period of time, suchas 24-72 hours, after which cells are assessed for cytotoxicity byviability, such as via use of Trypan Blue (Sigma, Dorset, UK) dyeexclusion and by propidium iodide (PI) and Annexin V staining(Invitrogen, Paisley UK) followed by FACS analysis.

Example 13: Cytotoxicity Analysis of Anti-CD22 scFv Fused to Amino AcidSubstituted PE in RAJI Cells

WT PE and amino acid substituted PE encoded by pIEX02-244 (SEQ ID NO:178) are fused to an anti-CD22 single-chain Fv (scFv). The VH and VL(V_(kappa)) regions of RFB4 (Campana D. et al., J. Immunol.,134:1524-1530 (1985); Mansfield, E., et al., Blood, 90:2020-2026 (1997)are synthesized. RFB4 VH is amplified using oligonucleotides: OL2043 andOL2044 (Table 12). RFB4 and VL (Vk) is amplified using oligonucleotidesOL2045 and OL2046 (Table 12). The RFB4 scFv is obtained using apull-through PCR reaction using oligonucleotides OL2047 introducing aNcoI site and OL2048 introducing a XhoI site. The resultant PCR productis subcloned into pET14b-K via NcoI and XhoI restriction enzymes(Fermentas catalog # FD0573 and FD0695, respectively).

The gene encoding RFB4 scFv is fused to genes encoding either WT PE oramino acid substituted PE encoded by pIEX02-244 (SEQ ID NO: 178) havinga C-terminal 8×His-tag followed by the sorting signal EDLK to givefusion sequences SEQ ID NO:184 and SEQ ID NO: 186, respectively, whichare cloned into the expression vector pET14b-K by fusion PCR. To createthese RFB4-PE fusions a fusion PCR is carried out. The RFB4 scFv gene isamplified from pET14b-RFB4 using oligonucleotides OL2318 introducing aNcoI site and OL2320 (Table 12). The WT PE or the lead amino acidsubstituted PE genes are amplified from pET14b-K-WT PE or pET14b-K-244PE, respectively, using oligonucleotides OL2321 and OL2322 introducing aN-terminal 8×His-EDLK and a XhoI site (Table 12). Both scFv and PE genesare fused by performing a PCR with oligonucleotides OL2318 and OL2322(Table 12). The resulting full-length fragments are subcloned intopET14b-K using NcoI and XhoI restriction enzymes. Plasmids aretransformed into BL21(DE3) E. coli (EMD Millipore, Feltham, UK) andclones are inoculated into 2TY+Amp and grown overnight at 37° C. Two mlof overnight culture is inoculated into 350 ml 2TY+Amp media in a 1 Lflask and grown to OD_(600nm)=1 before addition of IPTG (Sigma) to 1 mM(final concentration). Cultures are grown overnight at 30 degrees C.overnight and centrifuged at 10000 rpm. Bacterial pellets are frozen at−80 until ready to use.

Pellets are defrosted on ice, extracted with 10 ml B-PER® BacterialProtein Extraction Reagent (PIERCE® Biotechnology, Rockford, Ill.;Thermo Scientific, Hemel Hempstead, UK) containing Lysozyme and DNaseI(both Thermo Scientific), and rotated for 1 hour at room temperature.Samples are then centrifuged at 10000 rpm and supernatants arediscarded. Each pellet is resuspended in 5 ml B-PER containing Lysozymeand DNaseI as above and extracted for an additional 30 min at roomtemperature. After centrifugation, pellets are pooled and washedsuccessively with Wash Buffer A (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1mM EDTA, 0.5 M urea and 1.0% Triton X-100), Wash Buffer B (Buffer A butwithout urea), and twice with Wash Buffer C (Buffer A but without ureaor Triton X-100). After final wash, insoluble pellets are stored at −80°C. cOmplete® mini-EDTA protease inhibitors (Roche Diagnostics Ltd.) areincluded at each step.

Pellets are resuspended in 10 ml Solubilisation Buffer (50 mM Tris-HClpH 8.0, 100 mM NaCl, 8 M Urea and 1 mM DTT). OD_(280nm) is determinedand the samples are diluted to approximately 1 mg/ml in SolubilisationBuffer. Protein samples are allowed to denature for 4 hours at roomtemperature and centrifuged at 10000 rpm to remove insoluble debris. 10ml of each solubilised protein samples is injected into a pre-soaked 12ml, 3K MWCO cut-off SLIDE-A-LYSER® Dialysis Cassette dialysis device(Thermo Scientific; PIERCE® Biotechnology, Inc., IL, USA), and dialyzedby placement overnight in a beaker containing 2.5 L Refolding Buffer A(50 mM Tris HCl pH 8.0, 100 mM NaCl, 5 mM reduced glutathione, 1 mMoxidised glutathione, 0.1 M arginine, 4 M urea). Dialysis buffer isreplaced with, in order, 2.5 L Refolding Buffer B (Buffer A with 2 Murea), 2.5 L Refolding Buffer C (Buffer A with 1 M urea) and 5 LRefolding Buffer D (Buffer A without urea or arginine) for a minimum of4 hours at each step.

Each sample is recovered from the dialysis cassette, buffer exchangedinto 50 mM 2-N-morpholino)ethanesulfonic acid (MES) pH 6.2 using PD10desalting columns (GE Healthcare, Little Chalfont, UK) and loaded onto a1 ml SP FF Anion Exchange column (GE Healthcare). Each column is washedwith 50 mM MES pH 6.2 before eluting using a linear 0M to 1M NaClgradient in 50 mM MES pH6.2. Protein-containing fractions are pooled andrun through a pre-equilibrated 16/60 Size Exclusion column (GEHealthcare) using 1×PBS as running buffer. Fractions containing the mainprotein peaks are collected, pooled and concentrated to approximately 1ml, filter sterilized and quantified.

For cytotoxicity analysis, Raji cells (ATCC, CCL-86) are propagated ingrowth medium (RPMI-1640, 10% FBS, 1% Pen/Strep) and harvested duringmid-log growth phase. Cells are diluted to 1×10⁵ cells/ml in growthmedium and 50 microliter aliquots are dispensed per well in whitewalled, clear bottom 96 well plates (CORNING® catalogue #3610, FISHERSCIENTIFIC®, Loughborough, UK). Each protein concentration (8×4-folddilutions from 500 nanograms/ml) is tested in triplicate wells, andcontrols containing cells or growth medium only are also included. Testprotein is diluted to 2× desired concentration in growth medium. 50microliters of the test protein dilutions or controls are added to theRaji cells and plates are incubated 72 hrs in a humidified cell cultureincubator (37° C., 5% CO₂). After incubation, plates are equilibrated atroom temperature for 10 min. CELLTITER-GLO® (PROMEGA® catalogue #G7571)is prepared according to manufacturer's instructions and 100 microlitersis added per well. Plates are incubated for 10 min before reading via aFLUOstar OPTIMA fluorescence plate reader (BMG Labtech Ltd., Aylesbury,UK)(also known as a fluorometer).

REFERENCES

-   Aida Y. & Pabst M. J., Journal of Immunological Methods, 132:191-195    (1990)-   Al-Dosari et al., AAPS Journal, 11(4):671-681 (2009).-   Allen et al. (Editor), Ansel's Pharmaceutical Dosage Forms and Drug    Delivery Systems, Lippincott Williams & Wilkins; 9^(th) Ed. (2011).-   Andre et al., Curr Gene Ther., 10(4):267-280 (2010).-   Antoniewski et al., Mol. Cell Biol. 14:4465 (1994).-   Ash et al. (Editor), Handbook of Pharmaceutical Additives, Third    Edition, Synapse Information Resources, Inc.; 3^(rd) Ed. (2007).-   Baker & Carr, Current Drug Safety, 5(4):1-6 (2010).-   Baker & Jones, Curr. Opin. Drug. Disc. Dev., 10(2):219-227 (2007).-   Bodles-Brakhop et al., Mol. Ther., 17(4):585-592 (2009).-   Brent et al., Cell, 43:729-736 (1985).-   Bryson et al., BioDrugs., 24(1):1-8 (2010).-   Campana D. et al., J. Immunol., 134:1524-1530 (1985).-   Canadian Patent No. 2,012,311.-   Chaudhary et al., Proc. Natl. Acad. Sci. USA, 87:308-312 (1990).-   Cherbas et. al., Genes Dev. 5:120-131 (1991).-   Chester et. al., Expert Rev. Clin. Immunol., 1(4): 549-559 (2006).-   Colosimo et al., Biotechniques, 29(2):314-8, 320-322 (2000).-   Crouch, et al., J. Immunol. Methods, 160(1):81-88 (1993).-   Curiel et al., Hum. Gene Ther. 3:147 (1992).-   D'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995).-   Donnelly et al., Drug Deliv. 17(4):187-207 (2010).-   European Patent No. 234,994B1.-   European Patent No. 461,809B1.-   Feigner et al., Proc. Natl. Acad. Sci USA. 84:7413 (1987).-   Feigner et al., Science 337:387 (1989).-   Golzio et al., Curr Gene Ther. 10(4):256-266 (2010).-   Hansen et al., J. Immunother. 33(3):297-304 (2011).-   Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, 2nd ed., ISBN 978-087969314-5 (1988).-   Harlow & Lane, Using Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, ISBN 0879695447 (1999).-   Hausman & Cooper, The Cell: A Molecular Approach, Washington, D.C.:    ASM Press. p. 51 (2004).-   Higuchi, Using PCR to Engineer DNA, PCR Technology: Principles and    Applications for DNA Amplification, H. Erlich, ed., Stockton Press,    Chapter 6, pp. 61-70 (1989).-   Hochuli et al., J Interferon Cytokine Res. 17 Suppl 1:S15-21 (1997).-   Holgate & Baker, Idrugs, 12(4):233-237 (2009).-   Hu et al., Int. J. Cancer, 127(9):2222-2229 (2010).-   Hutchinson et al., J. Biol. Chem. 255:6551 (1978).-   Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986).-   Hwang et al., Cell 48:129-136 (1987).-   Itoi et al. J. Neurosci. 31(16):6132-6139 (2011).-   Jaber & Baker, J. Pharm. Biomed. Anal. 43(4): 1256-61 (2007).-   Karzenowski et al., Biotechniques 39: 191-200 (2005).-   Klein et al., Curr. Opin. Biotechnol. 4(5):583-590 (1993).-   Kreitman et al., Leuk. Lymphoma Suppl. 2:82-86 (2011).-   Krieg et al., Nucl. Acids Res. 12, 7057-7070 (1984).-   Kuan et al., Int. J. Cancer 129(1):111-21 (2011).-   Kyte & Doolittle, J. Mol. Biol. 157(1):105-132 (1982).-   Lim, et al., Hematology 10(3):255-9 (2005).-   Mackey et al., Proc. Natl. Acad. Sci USA 85:8027 (1988).-   Mansfield, E., et al., Blood, 90:2020-2026 (1997).-   Mareeva et al., J. Immunol. Methods 353(1-2):78-86 (2010).-   Miller et al., Somat. Cell Mol. Genet. 27(1-6): 115-34 (2002).-   Nagata et al., Adv. Drug Deliv. Rev. 61(11):977-985 (2009).-   Namaka, et al., Curr Med Res Opin. 22(2):223-39 (2006).-   Niles, et al., Anal. Biochem., 366, 197-206 (2007).-   O'Neil, “The Merck Index: An Encyclopedia of Chemicals, Drugs, and    Biologicals,” 14^(th) Ed. (2006).-   Oliphant et al., Gene 44:177 (1986).-   Olsson et al., J. Appl. Biochem 5, 347-445 (1983).-   Onda et al., Proc. Natl. Acad. Sci. USA 105(32):11311-11316 (2008).-   Onda et al., Proc. Natl. Acad. Sci. USA 108(14):5742-5747 (2011).-   Pastan et al., Leukemia and Lymphoma 52(S2):87-90 (2011).-   Pastan et al., Science 254:1173-1177 (1991).-   Pathak et al., Biotechnol J. 4(11):1559-1572 (2009).-   PDR Network, Physicians' Desk Reference 2011,” PDR Network (2010).-   PDR Network, Physicians' Desk Reference 2012,” PDR Network (2011).-   Pelham et al., Eur. J. Biochem. 67, 247-56 (1976).-   Perry et al., Drugs R D. 9(6):385-96 (2008).-   Pichon et al., Curr Opin Biotechnol. 21(5):640-645 (2010).-   PROMEGA® Technical Bulletin # TB126 (Revised December 2010).-   PROMEGA® Technical Bulletin # TB288 (Revised June 2009).-   PROMEGA® Technical Bulletin # TB288 (Revised August 2011).-   PROMEGA® Technical Bulletin # TB306 (Revised May 2009).-   PROMEGA® Technical Bulletin # TB359 (Revised May 2009).-   PROMEGA® Technical Bulletin # TB359 (Revised October 2011).-   PROMEGA® Technical Bulletin # TM045 (Revised May 2011).-   PROMEGA® Technical Bulletin # TM051 (Revised March 2009).-   PROMEGA® Technical Bulletin # TM051 (Revised September 2011).-   Rochlitz et al., “Gene therapy of cancer,” Swiss Med. Wkly.,    131(1-2):4-9 (2001).-   Rowe et al. (Editor), “Handbook of Pharmaceutical Excipients,”    Pharmaceutical Press, 6^(th) Ed. (2009).-   Sadowski et al., Nature 335:563 (1988).-   Schellekens et al., J. Interferon Cytokine Res. 17(Suppl. 1), S5-S8    (1997).-   Seetharam et al., Jour. Biol. Chem. 266:17376-17381 (1991).-   Shapira et al., Gastroenterology 140(3):935-946 (2011).-   Siegall et al., Mol. Cell. Biol. 10(6); 2443-2447 (1990).-   Siegall et al., Biochemistry 30:7154-7159 (1991).-   Squirrell et al., A Practical Guide to Industrial Uses of ATP    Luminescence in Rapid Microbiology, Cara Technology Ltd., Lingfield,    pp. 107-113 (1997).-   Stish et al., Br. J. Cancer 101(7):1114-1123 (2009).-   Theuer et al., J. Biol. Chem., 267(24): 16872-16877 (1992).-   Thomas et al., Clin. Cancer Res. 10:7079-7087 (2004).-   U.S. Patent Application Publication No. 2005/0228016.-   U.S. Patent Application Publication No. 2006/0100416.-   U.S. Patent Application Publication No. 2009/0123441.-   U.S. Patent Application Publication No. 2009/0142341.-   U.S. Patent Application Publication No. 2009/0298175.-   U.S. Pat. No. 4,892,827.-   U.S. Pat. No. 4,985,461.-   U.S. Pat. No. 5,117,057.-   U.S. Pat. No. 5,225,443.-   U.S. Pat. No. 5,324,637.-   U.S. Pat. No. 5,378,726.-   U.S. Pat. No. 5,459,127.-   U.S. Pat. No. 5,492,817.-   U.S. Pat. No. 5,530,028.-   U.S. Pat. No. 5,580,859.-   U.S. Pat. No. 5,583,024.-   U.S. Pat. No. 5,589,466.-   U.S. Pat. No. 5,641,641.-   U.S. Pat. No. 5,650,289.-   U.S. Pat. No. 5,674,713.-   U.S. Pat. No. 5,693,622;-   U.S. Pat. No. 5,700,673.-   U.S. Pat. No. 5,821,238.-   U.S. Pat. No. 6,013,836.-   U.S. Pat. No. 6,258,603.-   U.S. Pat. No. 6,602,677.-   U.S. Pat. No. 6,982,152.-   U.S. Pat. No. 7,083,911.-   U.S. Pat. No. 7,241,584.-   U.S. Pat. No. 7,282,348.-   U.S. Pat. No. 7,304,161.-   U.S. Pat. No. 7,304,162.-   U.S. Pat. No. 7,375,093-   U.S. Pat. No. 7,452,663.-   U.S. Pat. No. 7,456,315.-   U.S. Pat. No. 7,531,326.-   U.S. Pat. No. 7,563,879.-   U.S. Pat. No. 7,700,310.-   U.S. Pat. No. 7,732,128.-   U.S. Pat. No. 7,741,067.-   U.S. Pat. No. 7,750,136-   U.S. Pat. No. 7,919,269.-   U.S. Pat. No. 7,935,510.-   U.S. Pat. No. 8,076,517.-   U.S. Pat. No. 8,105,825.-   U.S. Pat. No. 8,854,044.-   Ulmer et al., Science 259:1745 (1993).-   University of the Sciences in Philadelphia (Editor), “Remington: The    Science and Practice of Pharmacy,” Lippincott Williams & Wilkins,    21^(st) Ed. (2005).-   Uskokovic et al., J. Biomed. Mater. Res. B Appl. Biomater    96(1):152-191 (2011).-   Wells, Cell Biol Toxicol. 26(1):21-28 (2010).-   Weldon & Pastan, FEBS Journal 278(23):4683-4700 (2011)-   WO 1995/018863 (PCT/FR1995/000022).-   WO 1995/021931 (PCT/FR1995/000098).-   WO 1996/017823 (PCT/FR1995/001595).-   WO 1996/025508 (PCT/FR1996/000248).-   WO 2001/070816 (PCT/US2001/090500).-   WO 2002/029075 (PCT/US2001/030608).-   WO 2002/066612 (PCT/US2002/005090).-   WO 2002/066613 (PCT/US2002/005090).-   WO 2002/066614 (PCT/U S/2002/005706).-   WO 2002/066615 (PCT/US2002/005708).-   WO 2003/027266 (PCT/US/2002/05234).-   WO 2003/027289 (PCT/US2002/005026).-   WO 2005/108617 (PCT/US2005/015089).-   WO 2008/073154 (PCT/US2007/016747).-   WO 2008/153801 (PCT/US2008/006757).-   WO 2009/025866 (PCT/US2008/010040).-   WO 2009/045370 (PCT/US2008/011270).-   WO 2009/048560 (PCT/US2008/011563).-   WO 2010/042189 (PCT/US2009/005510).-   WO 2011/119773 (PCT/US2011/029682).-   Wu et al., J. Biol. Chem. 263:14621 (1988).-   Wu et al., J. Biol. Chem. 267:963 (1992).-   Wu et al., J. Biol. Chem. 262:4429 (1987).-   Xiong et al., Pharmazie 66(3):158-64 (2011).-   Yao et al., Cell 71:63 (1992).-   Yao et al., Nature 366:476 (1993).-   Zielinski et al., J. Immunother. 32(8):817-825 (October-2009).-   Zoller et al., DNA 3:479 (1984).

1. A polypeptide having at least one Pseudomonas exotoxin A (PE-A)biological activity, wherein said polypeptide comprises one or moreamino acid substitutions compared to a wild-type PE-A polypeptide,wherein said one or more amino acid substitutions is a substitution of adifferent amino acid at one or more positions corresponding to aminoacid residues in the polypeptide of SEQ ID NO: 1, wherein saidsubstitution positions are selected from the group consisting of: a)isoleucine (I) at position 141; b) arginine (R) at position 146; c)glycine (G) at position 147; d) glutamine (Q) at position 149; e)asparagine (N) at position 150; f) threonine (T) at position 152; g)valine (V) at position 189; h) arginine (R) at position 192; i)glutamine (Q) at position 194; j) aspartic acid (D) at position 197; k)serine (S) at position 241; l) isoleucine (I) at position 321; and m)glutamine (Q) at position
 326. 2. The polypeptide of claim 1, whereinsaid one or more amino acid substitutions is a conservative amino acidsubstitution.
 3. The polypeptide of claim 1, wherein said one or moreamino acid substitutions is selected from the group consisting of: a)isoleucine (I) at position 141 is substituted with alanine (A),threonine (T), or histidine (H); b) arginine (R) at position 146 issubstituted with glutamine (Q) or alanine (A); c) glycine (G) atposition 147 is substituted with serine (S); d) glutamine (Q) atposition 149 is substituted with threonine (T); e) asparagine (N) atposition 150 is substituted with alanine (A); f) threonine (T) atposition 152 is substituted with alanine (A) or arginine (R); g) valine(V) at 189 is substituted with alanine (A); h) arginine (R) at position192 is substituted with alanine (A) or glutamine (Q); i) glutamine (Q)at position 194 is substituted with arginine (R); j) aspartic acid (D)at position 197 is substituted with lysine (K); k) serine (S) atposition 241 is substituted with threonine (T), asparagine (N), lysine(K), or proline (P); l) isoleucine (I) at position 321 is substitutedwith alanine (A), asparagine (N), histidine (H), threonine (T), orglutamine (Q); and m) glutamine (Q) at position 326 is substituted withglutamic acid (E).
 4. The polypeptide of claim 1, wherein saidpolypeptide comprises a substitution for isoleucine (I) at position 141,a substitution for threonine (T) at position 152, a substitution forarginine (R) at position 192, a substitution for aspartic acid (D) atposition 197, a substitution for serine (S) at position 241, and asubstitution for glutamine (Q) at position
 326. 5. The polypeptide ofclaim 1, wherein said polypeptide comprises a substitution of threonine(T) or alanine (A) for isoleucine (I) at position 141, a substitutionalanine (A) or arginine (R) for threonine (T) at position 152, asubstitution of alanine (A) for arginine (R) at position 192, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 6.The polypeptide of claim 1, wherein said polypeptide comprises asubstitution of threonine (T) for isoleucine (I) at position 141, asubstitution alanine (A) for threonine (T) at position 152, asubstitution of alanine (A) for arginine (R) at position 192, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 7.The polypeptide of claim 1, wherein said polypeptide comprises asubstitution of alanine (A) for isoleucine (I) at position 141, asubstitution alanine (A) for threonine (T) at position 152, asubstitution of alanine (A) for arginine (R) at position 192, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 8.The polypeptide of claim 1, wherein said polypeptide comprises asubstitution for isoleucine (I) at position 141, a substitution forthreonine (T) at position 152, a substitution for aspartic acid (D) atposition 197, a substitution for serine (S) at position 241, and asubstitution for glutamine (Q) at position
 326. 9. The polypeptide ofclaim 1, wherein said polypeptide comprises a substitution forisoleucine (I) at position 141, a substitution for threonine (T) atposition 152, a substitution for arginine (R) at position 192, asubstitution for aspartic acid (D) at position 197, and a substitutionfor serine (S) at position
 241. 10. The isolated polypeptide of claim 1,wherein said polypeptide comprises a substitution of alanine (A) orthreonine (T) for isoleucine (I) at position 141, a substitution ofarginine (R) or alanine (A) for threonine (T) at position 152, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 11.The isolated polypeptide of claim 1, wherein said polypeptide comprisesa substitution of alanine (A) for isoleucine (I) at position 141, asubstitution of arginine (R) for threonine (T) at position 152, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 12.The polypeptide of claim 1, wherein said polypeptide comprises asubstitution of alanine (A) for isoleucine (I) at position 141, asubstitution of alanine (A) for threonine (T) at position 152, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 13.The polypeptide of claim 1, wherein said polypeptide comprises asubstitution of threonine (T) for isoleucine (I) at position 141, asubstitution of alanine (A) for threonine (T) at position 152, asubstitution of lysine (K) for aspartic acid (D) at position 197, asubstitution of threonine (T) for serine (S) at position 241, and asubstitution of glutamic acid (E) for glutamine (Q) at position
 326. 14.The polypeptide of claim 1, wherein the at least one Pseudomonasexotoxin A (PE-A) biological activity comprises the ability to inhibitin vitro transcription/translation compared to a corresponding wild-typeor non-substituted PE-A polypeptide, wherein said ability to inhibit invitro transcription/translation is in an amount selected from the groupconsisting of: (a) at least 5% inhibition; (b) at least 10% inhibition;(c) at least 15% inhibition; (d) at least 20% inhibition; (e) at least25% inhibition; (f) at least 30% inhibition; (g) at least 40%inhibition; (h) at least 50% inhibition; (i) at least 60% inhibition;(j) at least 70% inhibition; (k) at least 80% inhibition; (l) at least90% inhibition; (m) at least 100% inhibition; (n) about 100% inhibition;and (o) 100% inhibition.
 15. The polypeptide of claim 1, comprising anumber of amino acid substitutions selected from the group consistingof: a) 1 amino acid substitution; b) 2 amino acid substitutions; c) 3amino acid substitutions; d) 4 amino acid substitutions; e) 5 amino acidsubstitutions; and f) 6 amino acid substitutions.
 16. The polypeptide ofclaim 1, wherein said polypeptide comprises one or more amino acidsubstitutions which prevent or reduce host immunogenic responsescompared to the same polypeptide without said one or more amino acidsubstitutions.
 17. The polypeptide of claim 16, wherein host immunogenicresponses are prevented or reduced in a human host.
 18. The polypeptideof claim 1, wherein the last five or six amino acids in said polypeptidecomprise one or more amino acid sequences selected from the groupconsisting of: (i) Arg-Glu-Asp-Leu-Lys; (ii) Arg-Glu-Asp-Leu; (iii)Lys-Asp-Glu-Leu; (iv) Glu-Asp-Leu-Lys; and (v) a dimer, trimer,pentamer, hexamer, septamer, or octamer of (i), (ii), or (iii), or anycombination thereof.
 19. The polypeptide of claim 1, wherein saidpolypeptide has one or more biological activities selected from thegroup consisting of: a) eukaryotic cell killing activity (cellcytotoxicity); b) inhibits translation elongation factor EF-2 biologicalactivity; c) induces or catalyzes ADP-ribosylation of EF-2; and d)inhibits protein synthesis.
 20. The polypeptide of claim 1, wherein saidone or more amino acid substitutions prevent or reduce host immunogenicresponses compared to the same polypeptide without the correspondingsaid one or more amino acid substitutions. 21.-56. (canceled)